U.S. patent number 5,677,274 [Application Number 08/082,849] was granted by the patent office on 1997-10-14 for anthrax toxin fusion proteins and related methods.
This patent grant is currently assigned to The Government of the United States as represented by the Secretary of. Invention is credited to Naveen Arora, Kurt R. Klimpel, Stephen H. Leppla, Peter J. Nichols, Yogendra Singh.
United States Patent |
5,677,274 |
Leppla , et al. |
October 14, 1997 |
**Please see images for:
( Certificate of Correction ) ** |
Anthrax toxin fusion proteins and related methods
Abstract
The present invention provides a nucleic acid encoding a fusion
protein comprising a nucleotide sequence encoding the anthrax
protective antigen (PA) binding domain of the native anthrax lethal
factor (LF) protein and a nucleotide sequence encoding an activity
inducing domain of a second protein. Also provided is a nucleic
acid encoding a fusion protein comprising a nucleotide sequence
encoding the translocation domain and LF binding domain of the
native anthrax PA protein and a nucleotide sequence encoding a
ligand domain which specifically binds a cellular target. Proteins
encoded by the nucleic acid of the invention, vectors comprising
the nucleic acids and hosts capable of expressing the protein
encoded by the nucleic acids are also provided. A composition
comprising the PA binding domain of the native LF protein
chemically attached to a non-LF activity inducing moiety is further
provided. A method for delivering an activity to a cell is
provided. The steps of the method include a) administering to the
cell a protein comprising the translocation domain and the LF
binding domain of the native PA protein and a ligand domain, and b)
administering to the cell a product comprising the PA binding
domain of the native LF protein and a non-LF activity inducing
moiety, whereby the product administered in step b) is internalized
into the cell and performs the activity within the cell. The
invention also provides proteins including an anthrax protective
antigen which has been mutated to replace the trypsin cleavage site
with residues recognized specifically by the HIV-1 protease.
Inventors: |
Leppla; Stephen H. (Bethesda,
MD), Klimpel; Kurt R. (Gaithersburg, MD), Arora;
Naveen (Delhi, IN), Singh; Yogendra (Delhi,
IN), Nichols; Peter J. (Welling Kent, GB) |
Assignee: |
The Government of the United States
as represented by the Secretary of (Washington, DC)
|
Family
ID: |
26694882 |
Appl.
No.: |
08/082,849 |
Filed: |
June 25, 1993 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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21601 |
Feb 12, 1993 |
5591631 |
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Current U.S.
Class: |
514/7.6 |
Current CPC
Class: |
C07K
14/005 (20130101); C07K 14/21 (20130101); C07K
14/32 (20130101); C07K 16/00 (20130101); C07K
19/00 (20130101); A61K 47/6425 (20170801); C12N
15/62 (20130101); A61K 38/00 (20130101); C07K
2319/00 (20130101); C07K 2319/55 (20130101); C12N
2740/13022 (20130101); C12N 2740/16222 (20130101); C07K
2317/77 (20130101) |
Current International
Class: |
A61K
47/48 (20060101); C07K 14/21 (20060101); C07K
14/195 (20060101); C07K 14/005 (20060101); C07K
14/16 (20060101); C07K 14/32 (20060101); C07K
16/00 (20060101); C07K 19/00 (20060101); C12N
15/62 (20060101); A61K 38/00 (20060101); A61K
039/00 () |
Field of
Search: |
;514/2 ;424/1.69 |
Other References
Klimpel et al, "Modified Anthrax Toxin is Cleaved . . . by HIV-1
Protease" J. Cell. Biochem, Suppl. 18B:163, abstract J515 (Jan.
1994). .
Friedlander, "Macrophages are Sensitive to Anthrax Lethal Toxin . .
. " J. Biol. Chem. 261(16):7123-7126 (Jun. 1986). .
Arora et al., J. Bio. Chem., 267(22):15542-15548 (1992). .
Arora et al. Abstract: Third Internatl. Sympos. on Immunotoxins,
Orlando, FL (Jun. 19-21, 1992). .
Quinn et al., J. Bio. Chem., 266(30):20124-20130 (1991). .
Oeltmann and Frankel, News, 5:2334-2337 (Jul. 1991). .
Singh et al., J. Bio. Chem., 266(23):15493-15497 (1991). .
Singh et al., J. Bio. Chem., 264(32):19103-19107 (1989). .
Leppla et al. Abstract: Fifth European Workshop on Bacterial
Protein Toxins, Veldhoven (Jun. 30-Jul. 5, 1991). .
Klimpel et al. Abstract: 1992 ASM General Meeting, New Orleans, LA
(May 1992). .
Novak, J.M., et al., "Functional Characterization of
Protease-treated Bacillus anthracis Protective Antigen," J. of
Biological Chemistry, 267(24):17186-17193 (Aug. 25, 1992). .
Ivins, B.E., et al., "Cloning and Expression of the Bacillus
anthracis Protective Antigen Gene in Bacillus subtilis," Infection
and Immunity, 54(2):537-542 (Nov. 1986). .
Molloy, S.S., et al, "Human Furin is a Calcium-dependent Serine
Endoprotease That Recognizes the Sequence Arg-X-X-Arg and
Efficiently Cleaves Anthrax Toxin Protective Antigen," J. of
Biological Chemistry, 267(23):16396-16402 (Aug. 15, 1992). .
Zhang, L., et al., "Inhibition of HIV-1 RNA Production by the
Diphtheria Toxin-Related IL-2 Fusion Proteins DAB.sub.486 IL-2 and
DAB.sub.389 IL-2," J. of Acquired Immune Deficiency Syndromes,
5(12):1181-1187 (1992). .
O'Hare, M., et al., "Cytotoxicity of a recombinant ricin-A-chain
fusion protein containing a proteolytically-cleavable spacer
sequence," FEBS Lett. 273(1,2):200-204 (Oct. 1990). .
Williams, D.P., et al., "Cellular Processing of the Interleukin-2
Fusion Toxin DAB.sub.486 -IL-2 and Efficient Delivery of Diphtheria
Fragment A to the Cytosol of Target Cells Requires Arg.sup.194," J.
of Biological Chemistry, 265(33):20673-20677 (Nov. 25, 1990). .
Ohishi, I., et al., "Visualizations of Binding and Internalization
of Two Nonlinked Protein Components of Botulinum C.sub.2 Toxin in
Tissue Culture Cells," Infection and Immunity, 60(11):4648-4655
(Nov. 1992). .
Arora, Navene., et al. (1992) "Potent hybrid cytotoxins of anthrax
lethal factor and the ADP-ribosylation domain of Pseudomonas
exotoxin A are translocated direct to the cytosol of mammalian
cells", Abstracts of the General Meeting of the American Society
for Microbiology, abstract n. B-33. .
Cataldi, Angel, et al. (1992) "Regulation of pag gene expression in
Bacillus anthracis: Use of a pag-lacZ transcriptional fusion", FEMS
Microbiology Letters, 98(1-3):89-94. .
Klimpel, Kurt R., et al. (1992) "Anthrax toxin protective antigen
is activated by a cell surface protease with the sequence
specificity and catalytic properties of furin", Proceedings of the
National Academy of Sciences of USA, 89(21):10277-10281. .
Arora, Navene, et al. (1993) "Residues 1-254 of anthrax toxin
lethal factor are sufficient to cause cellular uptake of fused
polypeptides", Journal of Biological Chemistry,
268(5):3334-3341..
|
Primary Examiner: Jagannathan; Vasu S.
Assistant Examiner: Romeo; David S.
Attorney, Agent or Firm: Townsend and Townsend and Crew
Parent Case Text
This application is in a continuation in part application of Ser.
No. 08/021,601 filed Feb. 12, 1993 now U.S. Pat. No. 5,591,631.
Claims
We claim:
1. A method for targeting compounds having a desired biological
activity not present on native anthrax lethal factor (LF) to a
specific cell population, comprising:
a) administering to the cell population a first compound comprising
a first protein consisting essentially of:
i) the translocation domain and the anthrax lethal factor (LF)
binding domain of the native anthrax protective antigen (PA)
protein, and
ii) a ligand domain that specifically binds the first protein to a
target on the surface of the cell population to bind the first
compound to said surface; and
b) administering to the resultant cell population a second compound
comprising a fusion protein or conjugate consisting essentially
of:
i) the anthrax protective antigen (PA) binding domain of the native
anthrax lethal factor (LF) protein, chemically attached to
ii) a biological activity-inducing polypeptide to bind the second
compound to the first compound on the surface of the cell
population, internalize the second compound into the cell
population, and effect the activity of the polypeptide therein.
2. A method according to claim 1, wherein the anthrax protective
antigen (PA) binding domain of said second compound comprises at
least the first 254 amino acid residues but less than all of the
amino acid residues of the anthrax lethal factor (SEQ. ID NO:
2).
3. A method according to claim 1, wherein the ligand domain of said
first compound is the ligand domain of the native anthrax
protective antigen (PA) protein.
4. A method according to claim 1, wherein said second compound
comprises the anthrax protective antigen (PA) binding domain of the
native anthrax lethal factor (LF) protein chemically attached to a
polypeptide through a peptide bond.
5. The method of claim 1, wherein the polypeptide of said second
compound is a toxin.
6. The method of claim 1, wherein the polypeptide of said second
compound is an enzyme.
7. The method of claim 1, wherein the ligand domain of said first
compound is an antibody.
8. The method of claim 1, wherein the ligand domain of said first
compound is a growth factor.
9. The method of claim 5, wherein the polypeptide of said second
compound is Pseudomonas exotoxin A (PE).
10. The method of claim 5, wherein the polypeptide of said second
compound is the A chain of Diptheria toxin.
11. The method of claim 5, wherein the polypeptide of said second
compound is shiga toxin.
12. The method of claim 7, wherein the ligand domain of said first
compound is a single chain antibody.
Description
BACKGROUND OF THE INVENTION
The targeting of cytotoxic or other moieties to specific cell types
has been proposed as a method of treating diseases such as cancer.
Various toxins including Diphtheria toxin and Pseudomonas exotoxin
A have been suggested as potential candidate toxins for this type
of treatment. A difficulty of such methods has been the inability
to selectively target specific cell types for the delivery of
toxins or other active moieties.
One method of targeting specific cells has been to make fusion
proteins of a toxin and a single chain antibody. A single-chain
antibody (sFv) consists of an antibody light chain variable domain
(V.sub.L) and heavy chain variable domain (V.sub.H), connected by a
short peptide linker which allows the structure to assume a
conformation capable of binding to antigen. In a diagnostic or
therapeutic setting, the use of an sFv may offer attractive
advantages over the use of a monoclonal antibody (MoAb). Such
advantages include more rapid tumor penetration with concomitantly
low retention in non-targeted organs (Yokota et al. Cancer Res
52:3402,1992), extremely rapid plasma and whole body clearance
(resulting in high tumor to normal tissue partitioning) in the
course of imaging studies (Colcher et al. Natl. Cancer Inst.
82:1191, 1990; Milenic et al. Cancer Res. 51:6363, 1991), and
relatively low cost of production and ease of manipulation at the
genetic level (Huston et al. Methods Enzymol. 203:46, 1991;
Johnson, S. and Bird, R. E. Methods Enzymol. 203:88, 1991). In
addition, sFv-toxin fusion proteins have been shown to exhibit
enhanced anti-tumor activity in comparison with conventional
chemically cross-linked conjugates (Chaudhary et al. Nature
339:394, 1989; Batra et al. Cell. Biol. 11:2200-2295, 1991). Among
the first sFv to be generated were molecules capable of binding
haptens (Bird et al. Science 242:423, 1988; Huston et al. Proc.
Natl. Acad. Sci. USA 85:5879, 1988), cell-surface receptors
(Chaudhary et al., 1989), and tumor antigens (Chaudhary et al.
Proc. Natl. Acad. Sci. USA 87:1066, 1990; Colcher et al.,
1990).
The gene encoding an sFv can be assembled in one of two ways: (i)
by de novo construction from chemically synthesized overlapping
oligonucleotides, or (ii) by polymerase chain reaction (PCR)-based
cloning of V.sub.L and V.sub.H genes from hybridoma cDNA. The main
disadvantages of the first approach are the considerable expense
involved in oligonucleotide synthesis, and the fact that the
sequence of V.sub.L and V.sub.H must be known before gene assembly
is possible. Consequently, the majority of the sFv reported to date
were generated by cloning from hybridoma cDNA; nevertheless, this
approach also has inherent disadvantages, because it requires
availability of the parent hybridoma or myeloma cell line, and
problems are often encountered when attempting to retrieve the
correct V region genes from heterologous cDNA. For example,
hybridomas in which the immortalizing fusion partner is derived
from MOPC-21 may express a V.sub.L kappa transcript which is
aberrantly rearranged at the VJ recombination site, and which
therefore encodes a non-functional light chain (Cabilly &
Riggs, 1985; Carroll et al., 1988). Cellular levels of this
transcript may exceed that generated from the productive V.sub.L
gene, so that a large proportion of the product on PCR
amplification of hybridoma cDNA will not encode a functional light
chain. A second disadvantage of the PCR-based method, frequently
encountered by the inventors, is the variable success of recovering
V.sub.H genes using the conditions so far reported in the
literature, presumably because the number of mismatches between
primers and the target sequence destabilizes the hybrid to an
extent which inhibits PCR amplification.
Thus, methods of targeting toxins to specific cells using
single-chain antibodies methods have been difficult to practice
because of the difficulties in obtaining single chain antibodies
and other targeting moieties. Also, none of the proposed treatment
methods has been fully successful, because of the need to fuse the
toxin to the targeting moiety, thus disrupting either the toxin
function or the targeting function. Thus, a need exists for a means
to target molecules having a desired activity to a specific cell
population.
Bacterial and plant protein toxins have evolved novel and efficient
strategies for penetrating to the cytosol of mammalian cells, and
this ability has been exploited to develop anti-tumor and anti-HIV
cytotoxic agents. Examples include ricin and Pseudomonas exotoxin A
(PE) chimeric toxins and immunotoxins.
Pseudomonas exotoxin A (PE) is a toxin for which a detailed
analysis of functional domains exists. The sequence is deposited
with GenBank. Structural determination by X-ray diffraction,
expression of deleted proteins, and extensive mutagenesis studies
have defined three functional domains in PE: a receptor-binding
domain (residues 1-252 and 365-399) designated Ia and Ib, a central
translocation domain (amino acids 253-364, domain II), and a
carboxyl-terminal enzymatic domain (amino acids 400-613, domain
III). Domain III catalyzes the ADP-ribosylation of elongation
factor 2 (EF-2), which results in inhibition of protein synthesis
and cell death. Recently it was also found that an extreme carboxyl
terminal sequence is essential for toxicity (Chaudhary et al. Proc.
Natl. Acad. Sci. U.S.A. 87:308-312, 1990; Seetharam et al. J. Biol.
Chem. 266:17376-17381, 1991). Since this sequence is similar to the
sequence that specifies retention of proteins in the endoplasmic
reticulum (ER) (Munro, S. and Pelham, H. R. B. Cell 48:899-907,
1987), it was suggested that PE must pass through the ER to gain
access to the cytosol. Detailed knowledge of the structure of PE
has facilitated use of domains II, Ib, and III (together designated
PE40) in hybrid toxins and immunotoxins.
Bacillus anthracis produces three proteins which when combined
appropriately form two potent toxins, collectively designated
anthrax toxin. Protective antigen (PA, 82,684 Da (Dalton) (SEQ ID
NOS: 3 and 4)) and edema factor (EF, 89,840 Da) combine to form
edema toxin (ET), while PA and lethal factor (LF, 90,237 Da (SEQ ID
NOS: 1 and 2)) combine to form lethal toxin (LT) (Leppla, S. H.
Alouf, J. E. and Freer, J. H., eds. Academic Press, London 277-302,
1991). ET and LT each conform to the AB toxin model, with PA
providing the target cell binding (B) function and EF or LF acting
as the effector or catalytic (A) moieties. A unique feature of
these toxins is that LF and EF have no toxicity in the absence of
PA, apparently because they cannot gain access to the cytosol of
eukaryotic cells.
The genes for each of the three anthrax toxin components have been
cloned and sequenced (Leppla, 1991). This showed that LF and EF
have extensive homology in amino acid residues 1-300. Since LF and
EF compete for binding to PA63, it is highly likely that these
amino-terminal regions are responsible for binding to PA63. Direct
evidence for this was provided in a recent mutagenesis study (Quinn
et al. J. Biol. Chem. 266:20124-20130, 1991); all mutations made
within amino acid residues 1-210 of LF led to decreased binding to
PA63. The same study also suggested that the putative catalytic
domain of LF included residues 491-776 (Quinn et al., 1991). In
contrast, the location of functional domains within the PA63
polypeptide is not obvious from inspection of the deduced amino
acid sequence. However, studies with monoclonal antibodies and
protease fragments (Leppla, 1991) and subsequent mutagenesis
studies (Singh et al. J. Biol. Chem. 266:15493-15497, 1991) showed
that residues at and near the carboxyl terminus of PA are involved
in binding to receptor.
PA is capable of binding to the surface of many types of cells.
After PA binds to a specific receptor (Leppla, 1991) on the surface
of susceptible cells, it is cleaved at a single site by a cell
surface protease, probably furin, to produce an amino-terminal
19-kDa fragment that is released from the receptor/PA complex
(Singh et al. J. Biol. Chem. 264:19103-19107, 1989). Removal of
this fragment from PA exposes a high-affinity binding site for LF
and EF on the receptor-bound 63-kDa carboxyl-terminal fragment
(PA63). The complex of PA63 and LF or EF enters cells and probably
passes through acidified endosomes to reach the cytosol.
Cleavage of PA occurs after residues 164-167, Arg-Lys-Lys-Arg(SEQ.
ID NO: 32). This site is also susceptible to cleavage by trypsin
and can be referred to as the trypsin cleavage site. Only after
cleavage is PA able to bind either EF or LF to form either ET or
LT.
Prior work had shown that the carboxyl terminal PA fragment (PA63)
can form ion conductive channels in artificial lipid membranes
(Blaustein et al. Proc. Natl. Acad. Sci. U.S.A. 86:2209-2213, 1989;
Koehler, T. M. and Collier, R. J. Mol. Microbiol. 5:1501-1506,
1991), and that LF bound to PA63 on cell surface receptors can be
artificially translocated across the plasma membrane to the cytosol
by acidification of the culture medium (Friedlander, A. M. J. Biol.
Chem. 261:7123-7126, 1986). Furthermore, drugs that block endosome
acidification protect cells from LF (Gordon et al. J. Biol. Chem.
264:14792-14796, 1989; Friedlander, 1986; Gordon et al. Infect.
Immun. 56:1066-1069, 1988). The mechanisms by which EF is
internalized have been studied in cultured cells by measuring the
increases in cAMP concentrations induced by PA and EF (Leppla, S.
H. Proc. Natl. Acad. Sci. U.S.A. 79:3162-3166, 1982; Gordon et al.,
1989). However, because assays of cAMP are relatively expensive and
not highly precise, this is not a convenient method of analysis.
Internalization of LF has been analyzed only in mouse and rat
macrophages, because these are the only cell types lysed by the
lethal toxin.
SUMMARY OF THE INVENTION
The present invention provides a nucleic acid encoding a fusion
protein comprising a nucleotide sequence encoding the PA binding
domain of the native LF protein and a nucleotide sequence encoding
an activity inducing domain of a second protein. Also provided is a
nucleic acid encoding a fusion protein comprising a nucleotide
sequence encoding the translocation domain and LF binding domain of
the native PA protein and a nucleotide sequence encoding a ligand
domain which specifically binds a cellular target. Proteins encoded
by the nucleic acid of the invention, vectors comprising the
nucleic acids and hosts capable of expressing the protein encoded
by the nucleic acids are also provided.
A composition comprising the PA binding domain of the native LF
protein chemically attached to an activity inducing moiety is
further provided.
A method for delivering an activity to a cell is provided. The
steps of the method include administering to the cell (a) a protein
comprising the translocation domain and the LF binding domain of
the native PA protein and a ligand domain and (b) a product
comprising the PA binding domain of the native LF protein and a
non-LF activity inducing moiety, whereby the product administered
in step (b) is internalized into the cell and performs the activity
within the cell.
Characteristics unique to anthrax toxin are exploited to make novel
cell-specific cytotoxins. A site in the PA protein of the toxin
which must be proteolytically cleaved for the activity-inducing
moiety of the toxin to enter the cell is replaced by the consensus
sequence recognized by a specific protease. Thus, the toxin will
only act on cells infected with intracellular pathogens which make
that specific protease.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the percent to which mutant proteins are
cleaved by purified HIV-1 protease. The mutant proteins include
protective antigen (PA) mutated to include the HIV-1 protease
cleavage site in place of the natural trypsin cleavage site.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Nucleic Acids
Lethal Factor (LF)
The present invention provides an isolated nucleic acid encoding a
fusion protein comprising a nucleotide sequence encoding the PA
binding domain of the native LF protein and a nucleotide sequence
encoding an activity inducing domain of a second protein. The LF
gene and native LF protein are shown in SEQ. ID NO: 1 and 2,
respectively. The PA gene and native PA protein are shown in SEQ.
ID NO: 3 and 4, respectively.
The second protein can be a toxin, for example Pseudomonas exotoxin
A (PE), the A chain of Diphtheria toxin or shiga toxin. The
activity inducing domains of numerous other known toxins can be
included in the fusion protein encoded by the presently claimed
nucleic acid. The activity inducing domain need not be a toxin, but
can have other activities, including but not limited to stimulating
or reducing growth, selectively inhibiting DNA replication,
providing enzymatic activity or providing a source of radiation. In
any case, the fusion proteins encoded by the nucleic acids of the
present invention must be capable of being internalized and capable
of expressing the specified activity in a cell. A given LF fusion
protein of the present invention can be tested for its ability to
be internalized and to express the desired activity using methods
as described herein, particularly in Examples 1 and 2.
An example of a nucleic acid of the invention comprises the
nucleotide sequence defined in the Sequence Listing as SEQ. ID NO:
5. This nucleic acid encodes a fusion of LF residues 1-254 with the
two-residue linker "TR" and PE residues 401-602 (SEQ. ID NO: 6).
The protein includes a Met-Val-Pro- sequence at the beginning of
the LF sequence. Means for obtaining this fusion protein are
further described below and in Example 1.
A further example of a nucleic acid of this invention comprises the
nucleotide sequence defined in the Sequence Listing as SEQ. ID NO:
7. This nucleic acid encodes a fusion of LF residues 1-254 with the
two-residue linker "TR" and PE residues 398-613. (SEQ. ID NO: 8)
The junction point containing the "TR" is the sequence LTRA and the
Met-Val-Pro- is also present. This fusion protein and methods for
obtaining it are further described below and in Example 2.
Another example of the nucleic acid of the present invention
comprises the nucleotide sequence defined in the Sequence Listing
as SEQ. ID NO: 9. This nucleic acid encodes a fusion of LF residues
1-254 with the two residue linker and PE residues 362-613. (SEQ. ID
NO: 10) This fusion protein is further described in Example 1.
Alternatively, the nucleic acid can include the entire coding
sequence for the LF protein fused to a non-LF activity inducing
domain. Other LF fusion proteins of various sizes and methods of
making and testing them for the desired activity are also provided
herein, particularly in Examples 1 and 2.
Protective Antigen (PA)
Also provided is an isolated nucleic acid encoding a fusion protein
comprising a nucleotide sequence encoding the translocation domain
and LF binding domain of the native PA protein and a nucleotide
sequence encoding a ligand domain which specifically binds a
cellular target.
An example of a nucleic acid of this invention comprises the
nucleotide sequence defined in the Sequence Listing as SEQ. ID NO:
11. This nucleic acid encodes a fusion of PA residues 1-725 and
human CD4 residues 1-178, the portion which binds to gp120 exposed
on HIV-1 infected cells (SEQ ID NO:12). This fusion protein and
methods for obtaining and testing fusion proteins are further
described below and in Examples 3, 4 and 5.
The PA fusion protein encoding nucleic acid provided can encode any
ligand domain that specifically binds a cellular target, e.g. a
cell surface receptor, an antigen expressed on the cell surface,
etc. For example, the nucleic acid can encode a ligand domain that
specifically binds to an HIV protein expressed on the surface of an
HIV-infected cell. Such a ligand domain can be a single chain
antibody which is expressed as a fusion protein as provided above
and in Examples 3, 4 and 5. Alternatively, the nucleic acid can
encode, for example, a ligand domain that is a growth factor, as
provided in Example 3.
Although the PA encoding sequence of the nucleic acid encoding the
PA fusion proteins of this invention need only include the
nucleotide sequence encoding the translocation domain and LF
binding domain of the native PA protein, the nucleic acid can
further comprise the nucleotide sequence encoding the remainder of
the native PA protein. Any sequences to be included beyond those
required, can be determined based on routine considerations such as
ease of manipulation of the nucleic acid, ease of expression of the
product in the host, and any effect on
translocation/internalization as taught in the examples.
Proteins
Proteins encoded by the nucleic acids of the present invention are
also provided.
LF Fusion Proteins
The present invention provides LF fusion proteins encoded by the
nucleic acids of the invention as described above and in the
examples. Specifically, fusions of the LF gene with domains II, Ib,
and III of PE can be made by recombinant methods to produce
in-frame translational fusions. Recombinant genes (e.g., SEQ ID
NOs: 5, 7 and 9) were expressed in Escherichia coli (E. coli), and
the purified proteins were tested for activity on cultured cells as
provided in Examples 1 and 2. Certain fusion proteins are
efficiently internalized via the PA receptor to the cytosol. These
examples demonstrate that this system can be used to deliver many
different polypeptides into targeted cells.
Although specific examples of these proteins are provided, given
the present teachings regarding the preparation of LF fusion
proteins, other embodiments having other activity inducing domains
can be practiced using routine skill.
Using current methods of genetic manipulation, a variety of other
activity inducing moieties (e.g., polypeptides) can be translated
as fusion proteins with LF which in turn can be internalized by
cells when administered with PA or PA fusion proteins. Fusion
proteins generated by this method can be screened for the desired
activity using the methods set forth in the Examples and by various
routine procedures. Based on the data presented here, the present
invention provides a highly effective system for delivery of an
activity inducing moiety into cells.
PA fusion proteins
The present invention provides PA fusion proteins encoded by the
nucleic acids of the invention. Specifically fusions of PA with
single chain antibodies and CD4 are provided.
Using current methods of genetic manipulation, a variety of other
ligand domains (e.g., polypeptides) can be translated as fusion
proteins with PA which in turn can specifically target cells and
facilitate internalization LF or LF fusion proteins. Based on the
data presented here, the present invention provides a highly
effective system for delivery of an activity inducing moiety into a
particular type or class of cells.
Although specific examples of these proteins are provided, given
the present teachings regarding the preparation of PA fusion
proteins, other embodiments having other ligand domains can be
practiced using routine skill. The fusion proteins generated can be
screened for the desired specificity and activity utilizing the
methods set forth in the example and by various routine procedures.
In any case, the PA fusion proteins encoded by the nucleic acids of
the present invention must be able to specifically bind the
selected target cell, bind LF or LF fusions or conjugates and
internalize the LF fusion/conjugate.
Conjugates
A composition comprising the PA binding domain of the native LF
protein chemically attached to an activity inducing moiety is
provided. Such an activity inducing moiety is an activity not
present on native LF. The composition can comprise an activity
inducing moiety that is, for example, a polypeptide, a
radioisotope, an antisense nucleic acid or a nucleic acid encoding
a desired gene product.
Using current methods of chemical manipulation, a variety of other
moieties (e.g., polypeptides, nucleic acids, radioisotopes, etc.)
can be chemically attached to LF and can be internalized into cells
and can express their activity when administered with PA or PA
fusion proteins. The compounds can be tested for the desired
activity and internalization following the methods set forth in the
Examples. For example, the present invention provides an LF protein
fragment 1-254 (LF1-254) with a cysteine residue added at the end
of LF1-254 (LF1-254Cys). Since there are no other cysteines in LF,
this single cysteine provides a convenient attachment point through
which to chemically conjugate other proteins or non-protein
moieties.
Vectors and Hosts
A vector comprising the nucleic acids of the present invention is
also provided. The vectors of the invention can be in a host
capable of expressing the protein encoded by the nucleic acid.
To express the proteins and conjugates of the present invention,
the nucleic acids can be operably linked to signals that direct
gene expression. A nucleic acid is "operably linked" when it is
placed into a functional relationship with another nucleic acid
sequence. For instance, a promoter or enhancer is operably linked
to a coding sequence if it affects the transcription of the
sequence. Generally, operably linked means that the nucleic acid
sequences being linked are contiguous and, where necessary to join
two protein coding regions, contiguous and in reading frame.
The gene encoding a protein of the invention can be inserted into
an "expression vector", "cloning vector", or "vector," terms which
usually refer to plasmids or other nucleic acid molecules that are
able to replicate in a chosen host cell. Expression vectors can
replicate autonomously, or they can replicate by being inserted
into the genome of the host cell. Vectors that replicate
autonomously will have an origin of replication or autonomous
replicating sequence (ARS) that is functional in the chosen host
cell(s). Often, it is desirable for a vector to be usable in more
than one host cell, e.g., in E. coli for cloning and construction,
and in a mammalian cell for expression.
The particular vector used to transport the genetic information
into the cell is also not particularly critical. Any of the
conventional vectors used for expression of recombinant proteins in
prokaryotic or eukaryotic cells can be used.
The expression vectors typically have a transcription unit or
expression cassette that contains all the elements required for the
expression of the DNA encoding a protein of the invention in the
host cells. A typical expression cassette contains a promoter
operably linked to the DNA sequence encoding the protein, and
signals required for efficient polyadenylation of the transcript.
The promoter is preferably positioned about the same distance from
the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
The DNA sequence encoding the protein of the invention can be
linked to a cleavable signal peptide sequence to promote secretion
of the encoded protein by the transformed cell. Additional elements
of the vector can include, for example, selectable markers and
enhancers. Selectable markers, e.g., tetracycline resistance or
hygromycin resistance, permit detection and/or selection of those
cells transformed with the desired DNA sequences (see, e.g., U.S.
Pat. No. 4,704,362).
Enhancer elements can stimulate transcription up to 1,000 fold from
linked homologous or heterologous promoters. Many enhancer elements
derived from viruses have a broad host range and are active in a
variety of tissues. For example, the SV40 early gene enhancer is
suitable for many cell types. Other enhancer/promoter combinations
that are suitable for the present invention include those derived
from polyoma virus, human or murine cytomegalovirus, the long
terminal repeat from various retroviruses such as murine leukemia
virus, murine or Rous sarcoma virus, and HIV. See, Enhancers and
Eukaryotic Expression, Cold Spring Harbor Pres, Cold Spring Harbor,
N.Y. 1983, which is incorporated herein by reference.
In addition to a promoter sequence, the expression cassette should
also contain a transcription termination region downstream of the
structural gene to provide for efficient termination. The
termination region can be obtained from the same gene as the
promoter sequence or can be obtained from a different gene.
For more efficient translation in mammalian cells of the mRNA
encoded by the structural gene, polyadenylation sequences are also
commonly added to the vector construct. Two distinct sequence
elements are required for accurate and efficient polyadenylation:
GU or U rich sequences located downstream from the polyadenylation
site and a highly conserved sequence of six nucleotides, AAUAAA,
located 11-30 nucleotides upstream. Termination and polyadenylation
signals that are suitable for the present invention include those
derived from SV40, or a partial genomic copy of a gene already
resident on the expression vector.
The vectors containing the gene encoding the protein of the
invention are transformed into host cells for expression.
"Transformation" refers to the introduction of vectors containing
the nucleic acids of interest directly into host cells by well
known methods. The particular procedure used to introduce the
genetic material into the host cell for expression of the protein
is not particularly critical. Any of the well known procedures for
introducing foreign nucleotide sequences into host cells can be
used. It is only necessary that the particular procedure utilized
be capable of successfully introducing at least one gene into the
host cell which is capable of expressing the gene.
Transformation methods, which vary depending on the type of host
cell, include electroporation; transfection employing calcium
chloride, rubidium chloride calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection;
infection (where the vector is an infectious agent); and other
methods. See, generally, Sambrook et al., (1989) supra, and Current
Protocols in Molecular Biology, supra. Reference to cells into
which the nucleic acids described above have been introduced is
meant to also include the progeny of such cells.
There are numerous prokaryotic expression systems known to one of
ordinary skill in the art useful for the expression of the antigen.
E. coli is commonly used, and other microbial hosts suitable for
use include bacilli, such as Bacillus subtilus, and other
enterobacteriaceae, such as Salmonella, Serratia, and various
Pseudomonas species. One can make expression vectors for use in
these prokaryotic hosts; the vectors will typically contain
expression control sequences compatible with the host cell (e.g.,
an origin of replication, a promoter). Any number of a variety of
well-known promoters can be used, such as the lactose promoter
system, a tryptophan (Trp) promoter system, a beta-lactamase
promoter system, or a promoter from phage lambda. The promoters
will typically control expression, optionally with an operator
sequence, and have ribosome binding site sequences, for example,
for initiating and completing transcription and translation. If
necessary, an amino terminal methionine can be provided by
insertion of a Met codon 5' and in-frame with the codons for the
protein. Also, the carboxy-terminal end of the protein can be
removed using standard oligonucleotide mutagenesis procedures, if
desired.
Host bacterial cells can be chosen that are mutated to be reduced
in or free of proteases, so that the proteins produced are not
degraded. For Bacillus expression systems in which the proteins are
secreted into the culture medium, strains are available that are
deficient in secreted proteases.
Mammalian cell lines can also be used as host cells for the
expression of polypeptides of the invention. Propagation of
mammalian cells in culture is per se well known. See, Tissue
Culture, Academic Press, Kruse and Patterson, ed. (1973). Host cell
lines may also include such organisms as bacteria (e.g., E. coli or
B. subtills), yeast, filamentous fungi, plant cells, or insect
cells, among others.
Purification of Protein
After standard transfection or transformation methods are used to
produce prokaryotic, mammalian, yeast, or insect cell lines that
express large quantities of the protein of the invention, the
protein is then purified using standard techniques which are known
in the art. See, e.g., Colley et al. (1989) J. Biol. Chem. 64:
17619-17622; and Methods in Enzymology, "Guide to Protein
Purification", M. Deutscher, ed. Vol. 182 (1990).
Standard procedures of the art that can be used to purify proteins
of the invention include ammonium sulfate precipitation, affinity
and fraction column chromatography, gel electrophoresis and the
like. See, generally, Scopes, R., Protein Purification,
Springer-Verlag, N.Y. (1982), and U.S. Pat. No. 4,512,922
disclosing general methods for purifying protein from recombinantly
engineered bacteria.
If the expression system causes the protein of the invention to be
secreted from the cells, the recombinant cells are grown and the
protein is expressed, after which the culture medium is harvested
for purification of the secreted protein. The medium is typically
clarified by centrifugation or filtration to remove cells and cell
debris and the proteins can be concentrated by adsorption to any
suitable resin such as, for example, CDP-Sepharose,
asialoprothrombin-Sepharose 4B, or Q Sepharose, or by use of
ammonium sulfate fractionation, polyethylene glycol precipitation,
or by ultrafiltration. Other means known in the art are equally
suitable. Further purification of the protein can be accomplished
by standard techniques, for example, affinity chromatography, ion
exchange chromatography, sizing chromatography, or other protein
purification techniques used to obtain homogeneity. The purified
proteins are then used to produce pharmaceutical compositions, as
described below.
Alternatively, vectors can be employed that express the protein
intracellularly, rather than secreting the protein from the cells.
In these cases, the cells are harvested, disrupted, and the protein
is purified from the cellular extract, e.g., by standard methods.
If the cell line has a cell wall, then initial extraction in a low
salt buffer may allow the protein to pellet with the cell wall
fraction. The protein can be eluted from the cell wall with high
salt concentrations and dialyzed. If the cell line glycosolates the
protein, then the purified glycoprotein may be enhanced by using a
Con A column. Anion exchange columns Mono Q.RTM. Pharmacia) and gel
filtration columns may be used to further purify the protein. A
highly purified preparation can be achieved at the expense of
activity by denaturing preparative polyacrylamide gel
electrophoresis.
Protein analogs can be produced in multiple conformational forms
which are detectable under nonreducing chromatographic conditions.
Removal of those species having a low specific activity is
desirable and is achieved by a variety of chromatographic
techniques including anion exchange or size exclusion
chromatography.
Recombinant analogs can be concentrated by pressure dialysis and
buffer exchanged directly into volatile buffers (e.g.,
N-ethylmorpholine (NEM), ammonium bicarbonate, ammonium acetate,
and pyridine acetate). In addition, samples can be directly
freeze-dried from such volatile buffers resulting in a stable
protein powder devoid of salt and detergents. In addition,
freeze-dried samples of recombinant analogs can be efficiently
resolubilized before use in buffers compatible with infusion (e.g.,
phosphate buffered saline). Other suitable buffers might include
hydrochloride, hydrobromide, sulphate acetate, benzoate, malate,
citrate, glycine, glutamate, and aspartate.
Specific Embodiments
Toxins Modified to Contain Intracellular Pathogen Protease
Recognition Sites
One aspect of the invention exploits the fact that PA and other
toxins must be proteolytically cleaved in order to acquire
activity, in conjunction with the fact that some cells infected
with an intracellular pathogen possess an active protease that has
a relatively narrow substrate specificity (for example,
HIV-infected cells). The protease site found in the native toxin is
replaced with an intracellular pathogen specific protease site.
Thus, the protease in cells that are infected by the intracellular
pathogen cleaves the modified toxin, which then becomes active and
kills the cell.
Intracellular pathogens that can be targeted by the products and
methods of the present invention include any pathogen that produces
a protease having a specific recognition site. Such pathogens can
include prokaryotes (including rickettsia, Mycobacterium
tuberculosis, etc.), mycoplasma, eukaryotic pathogens (e.g.
pathogenic fungi, etc.), and viruses. One example of an
intracellular pathogen that produces a specific protease is human
immunodeficiency virus (HIV). The HIV-1 protease cleaves viral
polyproteins to generate functional structural proteins as well as
the reverse transcriptase and the protease itself. HIV-1
replication and viral infectivity are absolutely dependent on the
action of the HIV-1 protease.
An intracellular pathogen specific protease site can be introduced
into any natural or recombinant toxin for which proteolytic
cleavage is required for toxicity. For example, one can replace the
anthrax PA trypsin cleavage site (R164-167) of PA with the HIV-1
protease site. Alternatively, the diphtheria toxin disulfide loop
sequence (see O'Hare, et al. FEBS 273 (1, 2): 200-204 (October
1990)) can be replaced with the HIV-1 protease cleavage site in
order to obtain a toxin specific to HIV-1 infected cells.
Similarly, the normally occurring diphtheria toxin sequence at
residues 191-194 (Williams, et al. J. Biol. Chem. 265(33):
20673-20677 (1990)) can be replaced by an intracellular pathogen
specific protease site such as the HIV-1 protease cleavage
sequence. The DAB486-IL-2 fusion toxin of Williams and the improved
DAB389-IL-2 toxin are effective on HIV-1 infected cells, which
express high levels of the IL-2 receptor. Williams, J. Biol. Chem.
265:20673. Addition of the HIV-1 protease cleavage site would
provide a further degree of specificity. Similarly, the botulinum
toxin C2 toxin is like the anthrax toxin in requiring a cleavage
within a native protein subunit (see Ohishi and Yanagimoto,
Infection and Immunity 60(11): 4648-4655 (November 1992)), so it
too can be made specific for cells infected by an intracellular
pathogen such as HIV-1.
In one embodiment of the invention, the protease site of PA is
replaced by the site recognized by the HIV-1 protease. The cellular
protease that cleaves PA absolutely requires the presence of the
Arg 164 and Arg 167 residues, because replacement of either residue
yields a PA molecule which is not cleaved after binding to the cell
surface. However, any PA substitution mutant which retains at least
one Arg or Lys residue within residues 164-167 can be activated by
treatment with trypsin. Because the PA63 fragments produced by
trypsin digestion have a variety of different amino terminal
residues, it is clear that there is not a strict constraint on the
identity of the terminal residues. Klimpel, et al. , Proc. Natl.
Acad. Sci. 89:10277-10281 (1992).
Replacement of residues 164-167 of PA with residues that match the
HIV-1 protease recognition site can render exogenously added PA
inactive on cells which do not possess the HIV-1 protease. However,
those cells that do express the HIV-1 protease (i.e., cells
infected with HIV-1 or cells engineered to produce the protease)
would cleave and thereby activate the mutant PA. The activated PA
proteins can then bind and internalize cytotoxic fusion proteins,
such as LF-PE, added exogenously.
Based on extensive studies of the substrate specificity of the
protease, several PA variants were designed and produced which
relate to the invention. These are shown below, with the residues
underlined between which the cleavage occurred. PA proteins which
have been mutated to replace R164-167 with an amino acid sequence
recognized by the HIV-1 protease are referred to as "PAHIV. " (SEQ.
ID NO: 13)
______________________________________ PAHIV#1 QVSQNYPIVQNI (SEQ ID
NO: 14) PAHIV#2 NTATIMMQRGNF (SEQ IDNO: 15) PAHIV#3 TVSFNFPQITLW
(SEQ ID NO: 16) PAHIV#4 GGSAFNFPIVMGG (SEQ ID NO: 17)
______________________________________
The mutant proteins PAHIV#(1-4) were cleaved correctly by the HIV-1
protease.
Table 1 shows the amino acids and their corresponding abbreviations
and symbols.
TABLE 1 ______________________________________ A Ala Alanine M Met
Methionine C Cys Cysteine N Asn Asparagine D Asp Aspartic acid P
Pro Proline E Glu Glutamic acid Q Gln Glutamine F Phe Phenylalanine
R Arg Arginine G Gly Glycine S Ser Serine H His Histidine T Thr
Threonine I Ile Isoleucine V Val Valine K Lys Lysine W Trp
Tryptophan L Leu Leucine Y Tyr Tyrosine
______________________________________
Preferably, the mutations at R164-167 of PA are accomplished by
cassette mutagenesis, although other methods are feasible as
discussed below. In summary, three pieces of DNA are joined
together. The first piece has vector sequences and encodes the
"front half" (5' end of the gene) of PA protein, the second is a
short piece of DNA (a cassette) and encodes a small middle piece of
PA protein and the third encodes the "back half" (3' end of the
gene) of PA. The cassette contains codons for the amino acids that
are required to complete the cleavage site for the intracellular
pathogen protease. This method was used to make mutants in the
plasmid pYS5 although other plasmids could be employed.
Alternatively, the mutations can be accomplished by use of the
polymerase chain reaction (PCR) and other methods as discussed
below. PCR duplicates a segment of DNA many times, resulting in an
amplification of that segment. The reaction produces enough of the
segment of DNA so that it can be modified with restriction enzymes
and cloned. During the reaction a synthetic oligonucleotide primer
is used to start the duplication of the target DNA segment. Each
synthetic primer can be designed to introduce novel DNA sequences
into the DNA molecule, or to change existing DNA sequences.
Modification of Toxins to Broaden or Alter Target Cell
Specificity
Another aspect of the invention involves compounds and methods for
broadening or changing the range of cell types against which a
toxin is effective. For example, the lethal anthrax toxin, PA+LF,
is acutely toxic to mouse macrophage cells, apparently due to the
specific expression in these cells of a target for the catalytic
activity of LF. Other cell types are not affected by LF. However,
in the present invention, LF is used to construct cytotoxins having
broad cell specificity.
A detailed analysis of the domains of LF identified the
amino-terminal 254 amino acids as the region that binds to PA63.
Fusion proteins containing residues 1-254 of LF and the
ADP-ribosylation domain of Pseudomonas exotoxin A (PE) were
designed according to the invention. These fusion proteins are
highly toxic to cultured cells, but only when PA is administered
simultaneously.
Synthesis of Genes that Encode Proteins of the Invention
Genes that encode toxins having altered protease recognition sites
or fusion proteins having a binding domain from one protein and an
activity inducing domain of a second protein can be synthesized by
methods known to those skilled in the art. As an example of
techniques that can be utilized, the synthesis of genes encoding
modified anthrax toxin subunits LF and PA are now described.
The DNA sequences for native PA and LF are known. Knowledge of
these DNA sequences facilitates the preparation of genes and can be
used as a starting point to construct DNA molecules that encode
mutants of PA and/or LF. The protein mutants of the invention are
soluble and include internal amino acid substitutions. Furthermore,
these mutants are purified from, or secreted from, cells that have
been transfected or transformed with plasmids containing genes
which encode these proteins. Methods for making modifications, such
as amino acid substitutions, deletions, or the addition of signal
sequences to cloned genes are known. Specific methods used herein
are described below.
The gene for PA or LF can be prepared by several methods. Genomic
and cDNA libraries are commercially available. Oligonucleotide
probes, specific to the desired gene, can be synthesized using the
known gene sequence. Methods for screening genomic and cDNA
libraries with oligonucleotide probes are known. A genomic or cDNA
clone can provide the necessary starting material to construct an
expression plasmid for the desired protein using known methods.
A protein encoding DNA fragment can be cloned by taking advantage
of restriction endonuclease sites which have been identified in
regions which flank or are internal to the gene. See Sambrook, et
al., Molecular Cloning: A Laboratory Manual 2d.ed. Cold Spring
Harbor Laboratory Press (1989), "Sambrook" hereinafter.
Genes encoding the desired protein can be made from wild-type genes
constructed using the gene encoding the full length protein. One
method for producing wild-type genes for subsequent mutation
combines the use of synthetic oligonucleotide primers with
polymerase extension on a mRNA or DNA template. This PCR method
amplifies the desired nucleotide sequence. U.S. Pat. Nos. 4,683,195
and 4,683,202 describe this method. Restriction endonuclease sites
can be incorporated into the primers. Genes amplified by PCR can be
purified from agarose gels and cloned into an appropriate vector.
Alterations in the natural gene sequence can be introduced by
techniques such as in vitro mutagenesis and PCR using primers that
have been designed to incorporate appropriate mutations.
The proteins described herein can be expressed intracellularly and
purified, or can be secreted when expressed in cell culture. If
desired, secretion can be obtained by the use of the native signal
sequence of the gene. Alternatively, genes encoding the proteins of
the invention can be ligated in proper reading frame to a signal
sequence other than that corresponding to the native gene. Though
the PA recombinant proteins of the invention are typically
expressed in B. anthracis, they can be expressed in other hosts,
such as E. coli.
The proteins of this invention are described by their amino acid
sequences and by their nucleotide sequence, it being understood
that the proteins include their biological equivalents such that
this invention includes minor or inadvertent substitutions and
deletions of amino acids that have substantially little impact on
the biological properties of the analogs. In some circumstances it
may be feasible to substitute rare or non-naturally occurring amino
acids for one or more of the twenty common amino acids listed in
Table 2. Examples include ornithine and acetylated or hydroxylated
forms. See generally Stryer, L., Biochemistry 3d ed. (1988).
Alternative nucleotide sequences can be used to express analogs in
various host cells. Furthermore, due to the degeneracy of the
genetic code, equivalent codons can be substituted to encode the
same polypeptide sequence. Additionally, sequences (nucleotide and
amino acid) with substantial identity to those of the invention are
also included. Identity in this sense means the same identity (of
base pair or amino acid) and order (of base pairs or amino acids).
Substantial identity includes entities that are greater than 80%
identical. Preferably, substantial identity refers to greater than
90% identity. More preferably, it refers to greater than 95%
identity.
Mutagenesis
Mutagenesis can be performed to yield point mutations, deletions,
or insertions to alter the specific regions of the genes described
above. Point mutations can be introduced by a variety of methods
including chemical mutagenesis, mutagenic copying methods and site
specific mutagenesis methods using synthetic oligonucleotides.
Cassette mutagenesis methods are conveniently used to introduce
point mutations into the specified regions of the PA or LF genes. A
double-stranded oligonucleotide region containing alterations in
the specified sequences of the gene is prepared. This
oligonucleotide cassette region can be prepared by synthesizing an
oligonucleotide with the sequence alteration in residues of the PA
or LF gene, annealing to a primer, elongating with the large
fragment of DNA polymerase and trimming with BstBI. This
double-stranded oligonucleotide is ligated into the Bamhi/BstBI
fragment from pYS5 and the PpuMI-BamHI fragment from pYS6 to
produce an intact recombinant DNA. Other methods of producing the
double stranded oligonucleotides and other recombinant DNA vectors
can be practiced.
Chemical mutagenesis can be performed using the M13 vector system.
A single strand M13 recombinant DNA is prepared containing
recombinant PA or LF DNA. Another M13 recombinant containing the
same recombinant DNA but in double stranded form is used to prepare
a deletion in the targeted region of the gene. This double stranded
M13 recombinant is cleaved into a linear molecule with an
endonuclease, denatured, and annealed with the single strand M13
recombinant, resulting in a single strand gap in the target region
of the PA or LF DNA.
This gapped DNA M13 recombinant is then treated with a compound
such as sodium bisulfite to deaminate the cytosine residues in the
single strand DNA region to uracil. This results in limited and
specific mutations in the single strand DNA region. Finally, the
gap in the DNA is filled in by incubation with DNA polymerase,
resulting in a U-A base pair to replace a G-C base pair in the in
unmutated portion of the gene. Upon replication the new recombinant
gene contains T-A base pairs, which are point mutations from the
original sequence. Other forms of chemical mutagenesis are also
available.
Mutagenic copying of the PA or LF recombinant DNA can be carried
out using several methods. For example, a single-stranded gapped
DNA region is created as described above. This region is incubated
with DNA polymerase I and one or more mutagenic analogs of normal
ribonucleoside triphosphates. Copying of the single stranded region
with the DNA polymerase substitutes the mutagenic analogs as the
single strand gap region is filled in. Transfection and replication
of the resulting DNA results in production of some mutated
recombinant DNAs for PA, LF, or EF which can then be selected by
cloning. Other mutagenic copying methods can be used.
Point mutations can be introduced into the specified regions of the
PA or LF genes by methods using synthetic oligonucleotides for
site-specific mutagenesis. PCR copying of the PA or LF genes is
performed using oligonucleotide primers covering the specified
target regions, and which contain modifications from the wild type
sequence in these regions. The PA gene in a pYS5 vector can be PCR
amplified using this method to result in mutations in the 164-167
position. PCR amplification can also be used to introduce mutations
in the target region of the LF gene.
Synthetic oligonucleotide methods of introducing point mutations
can be performed using heteroduplex DNA. A M13 recombinant DNA
vector containing the PA or LF gene is prepared and a
single-stranded M13 recombinant is produced. A single strand
oligonucleotide containing an alteration in the specified target
sequence for the PA or LF gene is annealed to the single strand M13
recombinant to produce a mismatched sequence. Incubation with DNA
polymerase I results in a double-stranded M13 recombinant
containing base pair mismatches in the specified region of the
gene. This M13 recombinant is replicated in a host such as B.
anthracis or E. coli to produce both wild type and mutant M13
recombinants. The mutated M13 recombinants are cloned and isolated.
Other vector systems for mutagenesis involving synthetic
nucleotides and heteroduplex formation can be applicable.
Expression of Proteins in Prokaryotic Cells
In addition to the use of cloning methods in bacteria such as
Bacillus anthracis for amplification of cloned sequences, it may be
desirable to express the proteins in other prokaryotes. It is
possible to recover a functional protein from E. coli transformed
with an expression plasmid encoding a PA or LF protein.
Conveniently, the mutated PA proteins of the invention were
expressed in B. anthracis and the LF-fusion proteins were expressed
in E. coli.
Methods for the expression of cloned genes in bacteria are well
known. See Sambrook. To optimize expression of a cloned gene in a
prokaryotic system, expression vectors can be constructed which
include a promoter to direct mRNA transcription termination. The
inclusion of selection markers in DNA vectors transformed in
bacteria are useful. Examples of such markers include the genes
specifying resistance to ampicillin, tetracycline, or
chloramphenicol.
See Sambrook, previously cited, for details concerning selection
markers and promoters for use in bacteria such as E. coli. In an
embodiment of this invention, pYS5 is a vector for the subcloning
and amplification of desired gene sequences although other vectors
could be used.
Strains of Bacillus Anthracis Producing Mutated Protein(s)
For PA protein production, B. anthracis strains cured of both pX01
and pX02 are preferred because they are avirulent. Examples of such
strains are UM23Cl-1 and UM44-1C9, obtained from Curtis Thorne,
University of Massachusetts. Similar strains can be made by curing
of plasmids, as described by P. Mikesell, et al. , "Evidence for
plasmid-mediated toxin production in Bacillus anthracis," Infect.
Immun. 39:371-376 (1983).
See generally commonly assigned U.S. patent application Ser. No.
08/042,745, filed Apr. 5, 1993, now abandoned incorporated by
reference herein.
Treatment Methods
A method for delivering a desired activity to a cell is provided.
The steps of the method include administering to the cell (a) a
protein comprising the translocation domain and the LF binding
domain of the native PA protein and a ligand domain, and (b) a
product comprising the PA binding domain of the native LF protein
and a non-LF activity inducing moiety, whereby the product
administered in step (b) is internalized into the cell and performs
the activity within the cell.
The method of delivering an activity to a cell can use a ligand
domain that is the receptor binding domain of the native PA
protein. Other ligand domains are selected for their specificity
for a particular cell type or class of cells. The specificity of
the PA fusion protein for the targeted cell can be determined using
standard methods and as described in Examples 2 and 3.
The method of delivering an activity to a cell can use an activity
inducing moiety that is a polypeptide, for example a growth factor,
a toxin, an antisense nucleic acid, or a nucleic acid encoding a
desired gene product. The actual activity inducing moiety used will
be selected based on its functional characteristics, e.g. its
activity.
A method of killing a tumor cell in a subject is also provided. The
steps of the method can include administering to the subject a
first fusion protein comprising the translocation domain and LF
binding domain of the native PA protein and a tumor cell specific
ligand domain in an amount sufficient to bind to a tumor cell. A
second fusion protein is also administered wherein the protein
comprises the PA binding domain of the native LF protein and a
cytotoxic domain of a non-LF protein in an amount sufficient to
bind to the first protein, whereby the second protein is
internalized into the tumor cell and kills the tumor cell.
The cytotoxic domain can be a toxin or it can be another moiety not
strictly defined as a toxin, but which has an activity that results
in cell death. These cytotoxic moieties can be selected using
standard tests of cytotoxicity, such as the cell lysis and protein
synthesis inhibition assays described in the examples.
The invention further provides a method of killing HIV-infected
cells in a subject. The method comprises the steps of administering
to the subject a first fusion protein comprising the translocation
domain and LF binding domain of the native PA protein and a ligand
domain that specifically binds to an HIV protein expressed on the
surface of an HIV-infected cell, in an amount sufficient to bind to
an HIV-infected cell. The next step is administering to the subject
a second fusion protein comprising the PA binding domain of the
native LF protein and a cytotoxic domain of a non-LF protein, in an
amount sufficient to bind to the first protein, whereby the second
protein is internalized into the HIV-infected cell and kills the
HIV-infected cell, thereby preventing propagation of HIV.
Although certain of the methods of the invention have been
described as using LF fusion proteins, it will be understood that
other LF compositions having chemically attached activity inducing
moieties can be used in the methods.
The fusion proteins and other compositions of the inventions can be
administered by various methods, e.g., parenterally,
intramuscularly or intrapertioneally.
The amount necessary can be deduced from other receptor/ligand or
antibody/antigen therapies. The amount can be optimized by routine
procedures. The exact amount of such LF and PA compositions
required will vary from subject to subject, depending on the
species, age, weight and general condition of the subject, the
severity of the disease that is being treated, the particular
fusion protein of composition used, its mode of administration, and
the like. Generally, dosage will approximate that which is typical
for the administration of cell surface receptor ligands, and will
preferably be in the range of about 2 .mu.g/kg/day to 2
mg/kg/day.
Depending on the intended mode of administration, the compounds of
the present invention can be in various pharmaceutical
compositions. The compositions will include, as noted above, an
effective amount of the selected protein in combination with a
pharmaceutically acceptable carrier and, in addition, can include
other medicinal agents, pharmaceutical agents, carriers, adjuvants,
diluents, etc. By "pharmaceutically acceptable" is meant a material
that is not biologically or otherwise undesirable, i.e., the
material can be administered to an individual along with the fusion
protein or other composition without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the pharmaceutical composition in which
it is contained.
Parenteral administration, if used, is generally characterized by
injection. Injectables can be prepared in conventional forms,
either as liquid solutions or suspensions, solid forms suitable for
solution or suspension in liquid prior to injection, or as
emulsions. A more recently revised approach for parenteral
administration involves use of a slow release or sustained release
system, such that a constant level of dosage is maintained. See,
e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference
herein.
Formulations and Administration
Proteins of the invention such as PAHIV are typically mixed with a
physiologically acceptable fluid prior to administration to a
mammal such as a human. Examples of physiologically acceptable
fluids include saline solutions such as normal saline, Ringer's
solution, and generally mixtures of various salts including
potassium and phosphate salts with or without sugar additives such
as glucose. The proteins are administered parenterally with
intravenous administration being the most typical route. Either a
bolus of the protein in solution or a slow infusion can be
administered intravenously. The choice of a bolus or an infusion
depends on the kinetics, including the half-life, of the protein in
the patient. An appropriate evaluation of the time for delivery of
the protein is well within the skill of the clinician.
Patients selected for treatment with PAHIV are infected with HIV-1
and they may or may not be symptomatic. Optimally, the protein
would be administered to an HIV-1 infected person who is not yet
symptomatic. The dosage range of a protein of the invention such as
PAHIV is typically from about 5 to about 25 micrograms per kilogram
of body weight of the patient. Usually, the dose is about 10
micrograms per kilogram of body weight of the patient. The dosage
is repeated at regular intervals, such as weekly for about 4 to 6
weeks. At that time the clinician may opt to evaluate the patient's
immune status, including immuno-tolerance to the PAHIV, to decide
future treatment.
The foregoing description and the following examples are offered
primarily for purposes of illustration. It will be readily apparent
to those skilled in the art that the operating conditions,
materials, procedural steps and other parameters of the system
described herein can be further modified or substituted in various
ways without departing from the spirit and scope of the invention.
For example, although human use has been discussed, veterinary use
of the invention is also feasible. For instance, cats suffer from a
so-called feline AIDS or feline immunodeficiency virus (FIV).
Protective antigen can be altered to include a protease cleavage
site specific for FIV. Thus, the invention is not limited by the
description and examples, but rather by the appended claims.
EXAMPLE 1
Fusions of Anthrax Toxin Lethal Factor to the ADP-Ribosylation
Domain of Pseudomonas Exotoxin
Reagents and General Procedures
Restriction endonucleases and DNA modifying enzymes were purchased
from GIBCO/BRL, Boehringer Mannheim, or New England Biolabs. Low
melting point agarose (Sea Plaque) was obtained from FMC Corp.
(Rockland, Me.). Oligonucleotides were synthesized on a PCR Mate
(Applied Biosystems) and purified on oligonucleotide purification
cartridges (Applied Biosystems). The PCR was performed with a DNA
amplification reagent (GeneAmp) from Perkin-Elmer Cetus Instruments
and a thermal cycler (Perkin-Elmer Cetus). The amplification
involved denaturation at 94.degree. C. for 1 min, annealing at
55.degree. C. for 2.5 min and extension at 72.degree. C. for 3 min,
for 30 cycles. A final extension was run at 72.degree. C. for 7
min. For amplification of PE fragments, 10% formamide was added in
the reaction mixture to decrease the effect of high GC content. DNA
sequencing reactions were done using the Sequenase version 1.0 from
U.S. Biochemical Corp. and DNA sequencing gels were made from Gel
Mix 6 from GIBCO/BRL. [.sup.35 S]deoxyadenosine
5'-[.alpha.-thio]triphosphate and L-[3,4,5-.sup.3 H]leucine were
purchased from Dupont-New England Nuclear. J774A.1 cells were
obtained from American Type Culture Collection. Chinese Hamster
Ovary (CHO) cells were obtained from Michael Gottesman (National
Cancer Institute, National Institutes of Health) (ATCC CCL 61).
Plasmid Construction
Construction of plasmids containing LF-PE fusions was performed as
follows. Varying portions of the PE gene were amplified by PCR,
ligated in frame to the 3' end of the LF gene, and inserted into
the pVEX115 f+T expression vector (provided by V. K. Chaudhary,
National Cancer Institute, National Institutes of Health). To
construct fusion proteins, the 3'-end of the native LF gene
(including codon 776 of the mature protein, specifying Ser) was
ligated with the 5'-ends of sequences specifying varying portions
of domains II, Ib, and III of PE. The LF gene was amplified from
the plasmid pLF7 (Robertson, D. L. and Leppla, S. H. Gene 44:71-78,
1986) by PCR using PCR using oligonucleotide primers which added
KpnI and MluI sites at the 5' and the 3' ends of the gene,
respectively. Similarly, varying portions of the PE gene (provided
by David FitzGerald, National Cancer Institute, National Institutes
of Health) were amplified by PCR so as to add MluI and EcORI sites
at the 5' and 3' ends. The PCR product of the LF gene was digested
with KpnI and the DNA was precipitated. The LF gene was
subsequently treated with MluI. Similarly, the PCR products of PE
amplification were digested with MluI and EcoRI. The expression
vector pVEX115 f+T was cleaved with KpnI and EcoRI separately and
dephosphorylated. This vector has a T7 promoter, OmpA signal
sequence, multiple cloning site, and T7 transcription terminator.
All the above DNA fragments were purified from low-melting point
agarose, a three-fragment ligation was carried out, and the product
transformed into E. coli DH5.alpha. (ATCC 53868). The four
constructs described in this report have the entire LF gene fused
to varying portions of PE. The identity of each construct was
confirmed by sequencing the junction point using a Sequenase kit
(U.S. Biochemical Corp. ). For expression, recombinant plasmids
were transformed into E. coli strain SA2821 (provided by Sankar
Adhya, National Cancer Institute, National Institutes of Health,
which is a derivative of BL21(.lambda.DE3) (Studier, F. W. and
Moffatt, B. A. J. Mol. Biol. 189:113-150, 1986). This strain has
the T7 RNA polymerase gene under control of an inducible lac
promotor and also contains the degP mutation, which eliminates a
major periplasmic protease (Strauch et al. J. Bacteriol.
171:2689-2696, 1989).
In the resulting plasmids, the LF-PE fusion genes are under control
of the T7 promoter and contain an OmpA signal peptide to obtain
secretion of the products to the periplasm so as to facilitate
purification. The design of the PCR linkers also led to insertion
of two non-native amino acids, Thr-Arg, at the LF-PE junction. The
four fusions analyzed in this report contain the entire 776 amino
acids of mature LF, the two added residues TR (Thr-Arg), and
varying portions of PE. In fusion FP33, the carboxyl-terminal end
of PE was changed from the native REDLK (Arg-Glu-Asp-Leu-Lys) (SEQ.
ID NO: 33) to LDER (SEQ. ID NO: 34), a sequence that fails to cause
retention in the ER (endoplasmic reticulum).
Expression and Purification of Fusion Proteins
Fusion proteins produced from pNA2, pNA4, pNA23 and pNA33 were
designated FP2, FP4, FP23 and FP33 respectively. E. coli strains
carrying the recombinant plasmids were grown in super broth (32 g/L
Tryptone, 20 g/L yeast extract, 5 g/L NaCl, pH 7.5) with 100
.mu.g/ml of ampicillin with shaking at 225 rpm at 37.degree. C. in
2-L cultures. When A.sub.600 reached 0.8-1.0,
isopropyl-1-thio-.beta.-D-galactopyranoside was added to a final
concentration of 1 mM, and cultures were incubated an additional 2
hr. EDTA and 1,10-o-phenanthroline were added to 5 mM and 0.1 mM
respectively, and the bacteria were harvested by centrifugation at
4000.times.g for 15 min at 4.degree. C. For extraction of the
periplasmic contents, cells were suspended in 75 ml of 20% sucrose
containing 30 mM Tris and 1 mM EDTA, incubated at 0.degree. for 10
min, and centrifuged at 8000.times.g for 15 min at 4.degree. C.
Cells were resuspended gently in 50 ml of cold distilled water,
kept on ice for 10 min, and the spheroplasts were pelleted. The
supernatant was concentrated with CENTRI PREP.RTM.-100 units
(Amicon) and loaded on a SEPHACRYL.RTM. S-200 (a cross-linked
co-polymer of alkyl dextron and N,N'-methylenebisacrylanide column
(40.times.2 cm) and 1 ml fractions were collected.
Fractions having full length fusion protein as determined by
immunoblots were pooled and concentrated as above. Protein was then
purified on an anion exchange column (Mono Q.RTM. HR5/5,
Pharmacia-LKB) using a NaCl gradient. The fusion proteins eluted at
280-300 mM NaCl. The proteins were concentrated again on
CENTRIPREP.RTM.-100 (Amicon Division) and the Mono Q.RTM.
chromatography was repeated. Protein concentrations were determined
by the bicinchoninic acid method (BCA Protein Assay Reagent,
Pierce), using bovine serum albumin as the standard. Proteins were
analyzed by polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulfate (SDS). Gels were either stained with
Coomassie Brilliant Blue or the proteins were electroblotted to
nitrocellulose paper which was probed with polyclonal rabbit
antisera to LF or PE (List Biological Laboratories, Campbell,
Calif). To determine the percent of full length protein, SDS gels
stained with Coomassie Brilliant Blue were scanned with a laser
densitometer (Pharmacia-LKB Ultrascan XL).
The proteins migrated during gel electrophoresis with molecular
masses of more than 106 kDa, consistent with the expected sizes,
and immunoblots confirmed that the products had reactivity with
antisera to both LF and PE. The fusion proteins differed in their
susceptibility to proteolysis as judged by the appearance of
smaller fragments on immunoblots, and this led to varying yields of
final product. Thus, from 2-L cultures the yields were FP2, 27
.mu.g; FP4, 87 .mu.g; FP23, 18 .mu.g; and FP33, 143 .mu.g.
Cell Culture Techniques and Protein Synthesis Inhibition Assay
CHO cells were maintained as monolayers in Eagle's minimum
essential medium (EMEM) supplemented with 10% fetal bovine serum,
10 mM 4-2(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
(pH 7.3), 2 mM glutamine, penicillin/streptomycin, and
non-essential amino acids (GIBCO/BRL). Cells were plated in 24- or
48-well dishes one day before the experiment. After overnight
incubation, the medium was replaced with fresh medium containing 1
.mu.g/ml of PA unless otherwise indicated. Fusion proteins were
added to 0.1-1000 ng/ml. All data points were done in duplicate.
Cells were further incubated for 20 hr at 37.degree. C. in 5%
CO.sub.2 atmosphere. The medium was then aspirated and cells were
incubated for 2 hr at 37.degree. C. with leucine-free medium
containing 1 .mu.Ci/ml [.sup.3 H]leucine. Cells were washed twice
with medium, cold 10% trichloroacetic acid was added for 30 min,
the cells were washed twice with 5% trichloroacetic acid and
dissolved in 0.150 ml 0.1M NaOH. Samples were counted in
Pharmacia-LKB 1410 liquid scintillation counter. In experiments to
determine if the toxin is internalized through acidified endosomes,
1 .mu.M monensin (Sigma) was added 90 min prior to toxin and was
present during all subsequent steps. To verify that the fusion
proteins were internalized through the PA receptor, competition
with native LF was carried out. PA (0.1 .mu.g/ml) and LF
(0.1-10,000 ng/ml) were added to the CHO cells to block the PA
receptor and the fusion proteins were added thereafter at
concentrations of 100 ng/ml for FP4 and FP23 and 5 ng/ml for FP 33.
Protein synthesis inhibition was measured after 20 hr as described
above.
Cytotoxic Activity of the Fusion Proteins
All four fusion proteins made and purified were toxic to CHO cells.
The concentration causing 50% lysis of cultured cells (EC.sub.50)
values of the proteins were 350, 8, 10, and 0.2 ng/ml for FP2, FP4,
FP23 and FP33 respectively (Table 1). These assays were done with
PA present at 1 ug/ml, exceeding the K.sub.m of 0.1 ug/ml (100 pM).
The fusion proteins had no toxicity even at 1 .mu.g/ml when PA was
omitted, proving that internalization of the fusion proteins was
occurring through the action of PA and the PA receptor. Native LF
has previously been shown to have no short-term toxic effects on
CHO cells when added with PA, and therefore was not included in
these assays. The fusion protein having only domain III and an
altered carboxyl-terminus (FP33) was most active, whereas the one
having the intact domains II and III and the native REDLK (SEQ. ID
NO: 33) terminus (FP2) was least active. The other two fusion
proteins (FP4 and FP23) had intermediate potencies.
Among proteins having ADP-ribosylation activity, potencies
equalling or exceeding 1 pM have previously been found only for
native diphtheria and Pseudomonas toxins acting on selected cells
(Middlebrook, J. L. and Dorlan, R. B. Can. J. Microbiol.
23:183-189, 1977) and for fusion proteins of PE and diphtheria
toxin when tested on cells containing>100,000 receptors for the
ligand-recognition domain of the fusion (EGF, transferrin, etc. )
(Pastan, I. and FitzGerald, D. Science 254:1173-1177, 1991;
Middlebrook, et al. 1977). For CHO cells, the potency of FP33
(EC.sub.50 =2 pM) is higher than that of PE itself (EC.sub.50 =420
pM), even though CHO cells probably have similar numbers of
receptors for both PA and PE (approx. 5,000-20,000). If the
intracellular trafficking of native PE delivers less than 5% of the
molecules to the cytosol, then the 200-fold greater potency of FP33
suggests that the PA/LF system has an inherently high efficiency of
delivery to the cytosol.
A comparison of the potencies of the four fusion proteins shows
that inclusion of domain II decreases potency. Thus, the fusion
with the lowest potency, FP2, was the one containing intact domains
II, Ib, and III. In designing the fusion proteins, all or part of
PE domain II and Ib was included in several of the constructs
because it could not be assumed that the translocation functions
possessed by PA and LF would be able to correctly traffic PE domain
III to the cytosol. The combination of domains II, Ib, and III,
termed PE40, has been used in a large number of toxic hybrid
proteins, by fusion to growth factors, monoclonal antibodies, and
other proteins (Pastan et al. 1991; Oeltmann, T. N. and Frankel, A.
E. FASEB J. 5:2334-2337, 1991), and some of these fusions have
shown substantial potency. Domain II was found to be essential in
these hybrid proteins to provide a translocation function not
present in the receptor-binding domain to which it was fused. The
potency of many of these PE40 fusion proteins appears to require
that they be trafficked through the Golgi and ER and
proteolytically activated in the same manner as native PE, so as to
achieve delivery of domain III to the cytosol. The fact that
inclusion of the entire domain II in the LF fusion protein FP2
instead decreased activity suggests that internalization of the LF
fusions occurs through a different route, one that does not easily
accommodate all the sequences in domain II.
Evidence that structures within PE residues 251-278 inhibit
translocation of the LF fusions comes from the 35-fold lower
potency of FP2 compared to FP23. One structure that might inhibit
translocation of the fusions is the disulfide loop formed by Cys265
and Cys287. In native PE, this disulfide loop appears to be
required for maximum activity. Thus, native PE and TGF-.alpha.-PE40
fusions become 10- to 100-fold less toxic if one or both these
cysteines are changed to serine. The disulfide loop probably acts
to constrain the polypeptide so that Arg276 and Arg279 are
susceptible to the intracellular protease involved in the cleavage
that precedes translocation. In contrast, the disulfide loop
decreases the potency of the LF fusions, perhaps by preventing the
unfolding needed for passage through a protein channel, thereby
acting in this situation as a "stop transfer" sequence. FP23, which
lacks Cys265, would not contain the domain II disulfide, and
therefore would not be subject to this effect. LF, like PA and EF,
contains no cysteines, and would not be prevented by disulfide
loops from the complete unfolding needed to pass through a protein
channel. The suggestion that disulfide loops act as stop-transfer
signals would predict that the disulfide Cys372-Cys379 in PE domain
Ib, which is retained in all four LF fusions would also decrease
potency. It should be noted that neither the fusions made here nor
the PE40 fusions have been analyzed chemically to determine if the
disulfides in domains II and III are actually formed. If the
disulfides do form correctly, it would be predicted that the
potencies of all of the fusion proteins, and especially that of
FP2, would be increased by treatment with reducing agents. These
analyses have not yet been performed. This analysis also suggests
that future LF fusions might be made more potent by omission of
domain Ib.
The other structural feature of PE known to affect intracellular
trafficking is the carboxyl terminal sequence, REDLK (SEQ. ID NO:
33), that specifies retention in the ER (Chaudhary et al. 1990;
Muro et al. 1987). To determine if the trafficking of the LF fusion
proteins was similar to that of PE, two of the fusion proteins were
designed so as to differ only in the terminal sequence. Replacement
of the native sequence by LDER (SEQ. ID NO: 34), one that does not
function as an ER retention signal, produced the most toxic of the
four fusion proteins, FP33. FP4, identical except that it retained
a functional REDLK (SEQ. ID NO: 33) sequence, was 30-fold less
potent. These data suggest that sequestration of the REDLK-ended
(SEQ. ID NO: 33) fusions decreased their access to cytosolic EF-2.
The implication is that PE may require the REDLK (SEQ. ID NO: 33)
terminus to be delivered to the ER for an obligatory processing
step, but then be limited in its final toxic potential by
sequestration from its cytosolic target. Finally, this comparison
strongly argues that internalization of the LF fusions does not
follow the same path as PE.
In designing the fusion proteins described here it was hoped that
they would have cytotoxic activity against cells that are
unaffected by anthrax lethal toxin, and this was successfully
realized as shown by the data obtained with CHO cells. However,
prior knowledge about LF did not provide a basis for predicting
whether the constructs would retain toxicity toward mouse
macrophages, the only cells known to be rapidly killed by anthrax
lethal toxin. Macrophages are lysed by lethal toxin in 90-120
minutes, long before any inhibition of protein synthesis resulting
from ADP-ribosylation of EF-2 leads to decreases in membrane
integrity or viability. This kinetic difference made it possible to
test directly for LF action. As discussed above, the fusion
proteins purified to remove the -89-kDa LF species formed by
proteolysis were not toxic to J774A.1 macrophages. This shows that
attachment of a bulky group to the carboxyl terminus of LF
eliminates its normal toxic activity. In the absence of any assay
for the putative catalytic activity of LF, it is not possible to
determine the cause of the loss of LF activity. The inability of
the fusions to lyse J774A.1 cells also argues against proteolytic
degradation of the fusions either in the medium during incubation
with cells or after internalization.
An important result of the invention described here is the
demonstration that the anthrax toxin proteins constitute an
efficient mechanism for protein internalization into animal cells.
The high potency of the present fusion proteins argues that this
system is inherently efficient, as well as being amenable to
improvement. The high efficiency results in part from the apparent
direct translocation from the endosome, without a requirement for
trafficking through other intracellular compartments. In addition
to its efficiency, the system appears able to tolerate heterologous
polypeptides.
Macrophage Lysis Assay of Fusion Proteins
Fusion proteins were assayed for LF functional activity on J774A.1
macrophage cell line in the presence of 1 .mu.g/ml PA. One day
prior to use, cells were scraped from flasks and plated in 48-well
tissue culture dishes. For cytotoxicity tests, the medium was
aspirated and replaced with fresh medium containing 1 .mu.g/ml PA
and the LF fusion proteins, and the cells were incubated for 3 hr.
All data points were performed in duplicate. To measure the
viability of the treated cells,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
was added to the cells to a final concentration of 0.5 mg/ml, and
incubation was continued for an additional 45 min to allow the
uptake and oxidation of MTT by viable cells. Medium was aspirated
and replaced by 200 .mu.l of 0.5% SDS, 40 mM HCl, 90% isopropanol
and the plates were vortexed to dissolve the blue pigment. The MTT
absorption was read at 570 nm using a UVmax Kinetic Microplate
Reader (Molecular Devices Corp.).
The crude periplasmic extracts from which the fusion proteins were
purified caused lysis of J774A.1 macrophages when added with PA,
indicating the presence of active LF species, probably formed by
proteolysis of the fusion proteins. Purification removed this
activity, so that none of the final fusion proteins had this
activity. This result showed both that the purified proteins were
devoid of full size LF or active LF fragments, and that the lytic
activity of LF for macrophages is blocked when residues from PE are
fused at its carboxyl terminus.
ADP-Ribosylation Assays
For assaying ADP-ribosylation activity, the method of Collier and
Kandel (Collier, R. J. and Kandel, J. J. Biol. Chem. 246:1496-1503,
1971) was used with some modification. A wheat germ extract
enriched for EF-2 was used in the reaction. Briefly, in a 200-.mu.L
reaction assay, 20 .mu.L of buffer (500 mM Tris, 10 mM EDTA, 50 mM
dithiothreitol and 10 mg/ml bovine serum albumin) was mixed with 30
.mu.L of EF-2, 130 .mu.L of H.sub.2 O or sample, and 20 .mu.L of
[adenylate-.sup.32 P]NAD (0.4 .mu.Ci per assay, ICN Biochemicals)
containing 5 .mu.M of non-radioactive NAD. Samples were incubated
for 20 min at 23.degree. C., the reactions were stopped by adding 1
ml 10% trichloroacetic acid, and the precipitates were collected
and washed on GA-6 filters (Gelman Sciences). The filters were
washed twice with 70% ethanol, air dried, and the radioactivity
measured.
Table 1 shows that all the fusion proteins were equally capable of
ADP-ribosylation of EF-2. FP2, which had little cytotoxic activity
on CHO cells, still retained full ADP-ribosylation activity. It was
also found that treatment with urea and dithiothreitol under
conditions that activate the enzymatic activity of native PE,
caused no increase in the ADP-ribosylation activity of the fusion
proteins, suggesting that the proteins were not folded so as to
sterically block the catalytic site.
Effect of Mutant PA on LF-PE Activity
To verify that uptake of the fusion proteins requires PA, the
activity of the fusion proteins was measured in the presence of a
mutant PA which is apparently defective in internalization. This
mutant, PA-S395C, has a serine to cysteine substitution at residue
395 of the mature protein, and retains the ability to bind to
receptor, become proteolytically nicked, and bind LF, but is unable
to lyse macrophages. When PA-S395C was substituted for native PA in
combination with FP33, no inhibition of protein synthesis
inhibition was observed. Similar results were obtained when the
other three fusion proteins were tested in combination with
PA-S395C.
Effect of Monensin on Activity of the Fusion Proteins
To verify that internalization of the fusion proteins was occurring
by passage through acidified endosomes in the same manner as native
LF, the ability of monensin to protect cells was examined. Addition
of monensin to 1 .mu.M decreased the potency of FP33 by
>100-fold. Protection against the other three fusion proteins
exceeded 20-fold.
LF Block of LF-PE Fusion Activity
To further verify that the fusion proteins were internalized
through the PA receptor, CHO cells were incubated with PA and
different amounts of LF to block the receptor and the fusion
proteins were added thereafter. Protein synthesis inhibition assays
showed that native LF could competitively block LF-PE fusion
proteins in a concentration-dependent manner.
The present data suggest that the receptor-bound 63-kDa proteolytic
fragment of PA forms a membrane channel and that regions at or near
the amino-termini of LF and EF enter this channel first and thereby
cross the endosomal membrane, followed by unfolding and transit of
the entire polypeptide to the cytosol. This model differs from that
for diphtheria toxin in that the orientation of polypeptide
transfer is reversed. Since both EF and LF have large catalytic
domains, extending to near their carboxyl termini, it appears
probable that the entire polypeptide crosses the membrane. In the
LF fusion proteins, the attached PE sequences would be carried
along with the LF polypeptide in transiting the channel to the
cytosol. Thus, the PA63 protein channel must tolerate diverse amino
acid residues and sequences. The data presented is consistent with
the mechanism of direct translocation of the LF proteins to the
cytosol as suggested herein.
TABLE 1 ______________________________________ Cytotoxic and
catalytic activity of LF-PE fusion proteins ADP- Toxicity
Ribosylation Amino acid content (EC.sub.50).sup.b activity Protein
LF Linker PE (pM) ng/ml (relative)
______________________________________ PE none none 1-613 420 23
100.sup.c FP2 776 TR 251-613 2700 350 82 FP4 776 TR 362-613 65 8
105 FP23 776 TR 279-613 70 10 108 FP33 776 TR 362-612.sup.a 2 0.2
118 ______________________________________ .sup.a REDLK (SEQ ID NO:
33) at carboxyl terminus is changed to LDER (SEQ ID NO: 34 ).
.sup.b Data is from this example, except for native PE, which is
from dat not shown, and is equal to a value previously reported
(Moehring, T. J. and Moehring, J. M. Cell 11:447-454, 1977). .sup.c
ADPribosylation was measured using 30 ng of fusion protein in a
final volume of 0.200 ml with 5 .mu.M NAD. Results were corrected
for the molecular weights of the proteins and normalized to PE.
EXAMPLE 2
Residues 1-254 of Anthrax Toxin Lethal Factor are Sufficient to
Cause Cellular Uptake of Fused Polypeptides Reagents and General
Procedures
Restriction endonucleases and DNA modifying enzymes were purchased
from GIBCO/BRL, Boehringer Mannheim or New England Biolabs. Low
melting point agarose (Sea Plaque) was obtained from FMC
Corporation. Oligonucleotides were synthesized on a PCR Mate
(Applied Biosystems) and purified with Oligonucleotide Purification
Cartridges (Applied Biosystems). Polymerase chain reactions (PCR)
were performed on a thermal cycler (Perkin-Elmer-Cetus) using
reagents from U. S. Biochemical Corp. or Perkin-Elmer-Cetus. DNA
was amplified as described in Example 1. The DNA was sequenced to
confirmed the accuracy of all of the constructs described in the
report. SEQUENASE version 2.0 from U.S. Biochemical Corp. was
utilized for the sequencing reactions, and DNA sequencing gels were
made with Gel Mix 8 from GIBCO/BRL. [.sup.35 S]dATP.alpha.S and
L-[3,4,5-.sup.3 H]leucine were purchased from Dupont-New England
Nuclear. Chinese hamster ovary cells (CHO) were obtained from
Michael Gottesman (NCI, NIH). J774A.1 macrophage cells were
obtained from American Type Culture Collection.
Plasmid Construction
Three types of LF protein constructs were made and analyzed in this
report. All the constructs were made by PCR amplification of the
desired sequences, using the native LF gene as template. LF
proteins deleted at the amino- or carboxyl-terminus were
constructed by a single PCR amplification reaction that added
restriction sites at the ends for incorporation of the construct
into the expression vector. LF proteins deleted for one or more of
the 19-amino acid repeats that comprise residues 308-383 were
constructed by ligating the products of two separate PCR reactions
that amplified the regions bracketing the deletion. The third group
of constructs were fusions of varying portions of the amino
terminus of LF with PE domains Ib and III. Like the
internally-deleted LF proteins, these LF-PE fusions were also made
by ligation of two separate PCR products. In the latter two types
of constructs, the ligation of the PCR products resulted in
addition of a linker, ACGCGT, at the junction points. This
introduced two non-native residues, Thr-Arg, between the fused
domains. The PCR manipulations also added three non-native amino
acids, Met-Val-Pro, as an extension to the native amino terminus on
all the constructs described in this report. Addition of this
sequence is not likely to alter the activity of the constructs
(discussed below). It should be noted that the LF-PE fusions
described herein contain this three-residue extension.
For PCR reactions to make deletions of 40 and 78 amino acids from
the amino-terminus of LF, two different mutagenic oligonucleotide
primers were made which were substantially identical to the LF gene
template at the intended new termini, and which added KpnI sites at
their 5'-ends. Another (non-mutagenic) oligonucleotide primer for
introduction of a BamHI site at the 3' end of LF was prepared.
Similarly, to make deletions at the carboxyl-terminus of LF, two
different mutagenic primers were used which truncated LF at
residues 729 and 693 and introduced a BamHI site next to the new 3'
ends of the LF gene. A second (non-mutagenic) oligonucleotide
primer specific for the amino terminus of LF was made which
introduced a KpnI site at the 5' end of the gene. All of the
primers noted above were used in PCR reactions on a pLF7 template
(Robertson and Leppla, 1986) to synthesize DNA fragments having
KpnI and BamHI sites at their 5' and 3' ends, respectively. The
amplified LF DNAs containing the amino- and carboxyl-terminal
deletions were digested with the appropriate restriction enzymes.
The expression vector pVEX115f+T (provided by V. K. Chaudhary, NCI,
NIH) was cleaved sequentially with KpnI and BamHI and
dephosphorylated. This expression vector contains a T7 promoter, an
OmpA signal sequence for protein transport to the periplasm, a
multiple cloning site that includes KpnI and BamHI sites, and a T7
transcription terminator. The LF and pVEX115f+T DNA fragments were
purified from low melting point agarose, ligated overnight, and
transformed into E. coli DH5.alpha.. Transformants were screened by
restriction digestion to identify the desired recombinant plasmids.
Proteins produced by these constructs are designated according to
the amino acid residues retained; for example the LF truncated at
residue 693 is designated LF.sup.1-693. All of the mutant LF
proteins described above contain three non-native amino acids,
Met-Val-Pro, added to the amino-terminus as a result of the PCR
manipulations.
To analyze the role of the repeat region of LF, four different
constructs were made: 1., removal of the entire repeat region
(LF.sup.1-307.TR.LF.sup.384-776), 2., removal of the first repeat
(LF.sup.1-307.TR.LF.sup.327-776) 3., removal of the last repeat
(LF.sup.1-364.TR.LF.sup.384-776), and 4., removal of repeats 2-4
(LF.sup.1-326.TR.LF.sup.384-776). To construct
LF.sup.1-307.TR.LF.sup.384-776, four different primers were used in
two separate PCR reactions. To amplify LF.sup.1-307, one
oligonucleotide primer was made at the 5'-end of the LF gene which
added a KpnI site, and a second primer was constructed at the end
of residue 307, introducing an MluI site. For amplifying
LF.sup.384-776, a third primer was made at residue 384 with an
added MluI site, and the fourth primer was made at the residue 776
which introduced a BamHI site at the end. Two PCR amplifications
were done using primers one/two and three/four with pLF7 as
template (Robertson and Leppla, 1986). The first amplification
reaction was digested with KpnI and MluI separately, and the second
amplification reaction was digested with MluI and BamHI. The
expression vector pVEX115f+T was digested separately with KpnI and
BamHI and dephosphorylated. All three fragments were gel purified,
ligated overnight at 16.degree. C. and transformed into E. coli
DH5.alpha.. The other three constructs were made by similar
strategies. Oligonucleotide primers one and four were the same for
all four constructs, whereas primers two and three were changed
accordingly. All four constructs contain Met-Val-Pro at the amino
terminus of LF and Thr-Arg at the site of the repeat region
deletion.
To construct LF-PE fusion proteins, fragments of the LF gene
extending from the amino terminus to various lengths were amplified
from plasmid pLF7 (Robertson and Leppla, 1986) by PCR using a
common oligonucleotide primer that added a KpnI site at the 5' end
and mutagenic primers which added MluI sites at the intended new 3'
ends. The PCR products of the LF gene were digested with KpnI, the
DNAs were precipitated, and subsequently digested with MluI.
Domains Ib and III of the PE gene (provided by David FitzGerald,
NCI, NIH) were amplified by PCR using primers which added MluI and
EcoRI sites at the 5' and 3' ends, respectively. The PCR product of
PE was digested with MluI and EcoRI. Similarly, the expression
vector pVEX115f+T was digested with KpnI and EcoRI. All DNA
fragments were purified from low-melting agarose gels,
three-fragment ligations were carried out, and the products were
transformed into E. coli DH5.alpha.. The three constructs described
in this example have 254, 198 and 79 amino acids of LF joined with
PE domains Ib and III. These fusion proteins are designated
LF.sup.1-254.TR.PE.sup.362-613 (SEQ. ID NO: 10),
LF.sup.1-198.TR.PE.sup.362-613, and LF.sup.1-79.TR.PE.sup.362-613,
respectively. The proteins retain the native carboxyl-terminal
sequence of PE, REDLK (SEQ. ID NO: 33). It should be noted that
these abbreviations do not specify the entire amino acid content of
the proteins, because all the constructs also contain Met-Val-Pro,
which was added to the amino-terminus of the LF domain by the PCR
manipulations.
Expression and Purification of Deleted LF and Fusion Proteins
Recombinant plasmids were transformed into E. coli SA2821 (provided
by Sankar Adhya, NCI, NIH), a derivative of BL21(.lambda.DE3)
(Studier and Moffatt, 1986) that lacks the proteases encoded by the
lon, OmpT, and degP genes, and has the T7 RNA polymerase gene under
control of the lac promoter (Strauch et al., 1989). Transformants
were grown in super broth with 100 .mu.g/ml ampicillin, with
shaking at 225 rpm, 37.degree. C., in 2-L cultures. When A.sub.600
reached 0.8-1.0, isopropyl-1-thio-.beta.-D-galactopyranoside was
added to a final concentration of 1 mM, and cultures were incubated
for an additional 2 h. EDTA and 1,10-o-phenanthroline were added to
5 and 0.1 mM, respectively, and periplasmic protein was extracted
as described in Example 1. The supernatant fluids were concentrated
by CENTRIPREP.RTM.-30 units (Amicon) and proteins were purified to
near homogeneity by gel filtration SEPHACRYL.RTM. S-200 (a
cross-linked co-polymer of alkyl dextron and
N,N,'-methylenebisacrylanide Pharmacia-LKB) and anion exchange
chromatography (Mono Q.RTM., Pharmacia-LKB) as described in Example
1. To determine the percentage of full length protein, SDS gels
stained with Coomassie Brilliant Blue were scanned with a laser
densitometer (Pharmacia-LKB Ultrascan XL). Western blots were
performed as described previously (Singh et al., 1991).
The LF proteins having terminal deletions and the LF-PE fusion
proteins were obtained from periplasmic extracts and purified to
near homogeneity by gel filtration and anion exchange
chromatography. The migration of the proteins was consistent with
their expected molecular weights. Immunoblots confirmed that the LF
proteins had reactivity with LF antisera, and the LF-PE fusion
proteins had reactivity with both LF and PE antisera. Fusion
proteins and terminally-deleted LF proteins differed in their
susceptibility to proteolysis as judged by the appearance of
peptide fragments on the immunoblots, and this was also reflected
in the different amounts of purified proteins obtained. Thus, from
2-L cultures the yields of purified proteins were LF.sup.41-776, 39
.mu.g; LF.sup.79-776, 32 .mu.g; LF.sup.1-729, 50 .mu.g;
LF.sup.1-693, 46 .mu.g; LF.sup.1-254.TR.PE.sup.362-613, 184 .mu.g;
LF.sup.1-198.TR.PE.sup.362-613, 80 .mu.g;
LF.sup.1-79.TR.PE.sup.362-613, 127 .mu.g.
LF proteins deleted in the repeat region were found to be unstable
and full size product could not be purified. Therefore, the
activities of these proteins were determined by assay of crude
periplasmic extracts, and immunoblots were used to estimate the
amount of the full size proteins present.
Cytotoxicity on Macrophages of LF Proteins Having Terminal and
Internal Deletions
Deleted LF proteins were assayed for LF functional activity on the
J774A.1 macrophage cell line in the presence of native PA as
described in Example 1. Briefly, cells were plated in 24- or
48-well dishes in Dulbecco's modified Eagle medium (DMEM)
containing 10% fetal bovine serum, and allowed to grow for 18 h. PA
(1 .mu.g/ml) and the mutant LF proteins were added and cells were
incubated for 3 h. To measure the viability of the treated cells,
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
was added to the cells to a final concentration of 0.5 mg/ml. After
incubating for 45 min, the medium was aspirated and cells were
dissolved in 90% isopropanol, 0.5% SDS, 40 mM HCl, and read at 540
nm using a UVmax Kinetic Microplate Reader (Molecular Devices
Corp.).
To determine the extent of essential sequences at the amino
terminus of LF, the toxicities of the two LF proteins deleted at
the amino-terminus were measured in combination with PA in the
macrophage lysis assay. Purified LF.sup.41-776 and LF.sup.79-776
were unable to lyse J774A.1 macrophage cells. This indicates that
some portion of the sequence preceding residue 41 is needed to
maintain an active LF protein.
To examine the role of the carboxyl terminus of LF, two proteins
truncated in this region were prepared and analyzed. The proteins
LF.sup.1-693 and LF.sup.1-729 were assayed on J774A.1 cells and
found to be inactive. This is presumed to be due to inactivation of
the putative catalytic domain.
To begin study of the role of the repeat region of LF, four
constructs were made having deletions in this region. The proteins
expressed from these mutants were unstable. Of the four deleted
proteins, only LF.sup.1-307.TR.LF.sup.327-776 had immunoreactive
material at the position expected of intact fusion protein. The
amount of intact LF.sup.1-307.TR.LF.sup.327-776 was similar to that
of native LF expressed in the same vector. When these unpurified
periplasmic extracts were tested in J774A.1 macrophages, only the
native LF control was toxic. LF.sup.1-307.TR.LF.sup.327-776 did not
lyse macrophages even when present at 50-fold higher concentration
than that of crude periplasmic protein of LF. Conclusions cannot be
drawn about the toxicities of the other three constructs because
full size fusion proteins were not present in the periplasmic
extracts.
Cell Culture Techniques and Protein Synthesis Inhibition Assay of
Fusion Proteins
CHO cells were maintained as monolayers in .alpha.-modified minimum
essential medium (.alpha.-MEM) supplemented with 5% fetal bovine
serum, 10 mM HEPES (pH 7.3), and penicillin/streptomycin. Protein
synthesis assays were carried out in 24- or 48-well dishes as
described in Example 1. CHO cells were incubated with PA (0.1
ug/ml) and varying concentrations of LF, which is expected to block
the receptor. Fusion proteins were added at fixed concentrations,
as follows: FP4, 100 ng/ml, FP23, 100 ng/ml, and FP33, 5 ng/ml.
Cells were incubated for 20 hr and protein synthesis inhibition was
evaluated by [.sup.3 H]leucine incorporation.
Cytotoxicity of the LF-PE Fusion Proteins on CHO Cells
The use of fusion proteins provides a more defined method for
measuring the translocation of LF, as demonstrated in Example 1
showing that fusions of LF with domains Ib and III of PE are highly
toxicy. Translocation of these fusions is conveniently measured
because domain III blocks protein synthesis by ADP-ribosylation of
elongation factor 2. The new fusions containing varying portions of
LF fused to PE domains Ib and III were designed to identify the
minimum LF sequence able to promote translocation. The EC.sub.50 of
LF.sup.1-254.TR.PE.sup.362-613 (SEQ. ID NO: 10) was 1.7 ng/ml,
whereas LF.sup.1-198.TR.PE.sup.362-613 and
LF.sup.1-79.TR.PE.sup.362-613 did not kill 50% of the cells even at
a 1200-fold higher concentration. Other constructs were also made
and analyzed, containing larger portions of LF fused to PE domains
Ib and III, and found those to be equal in potency to
LF.sup.1-254.TR.PE.sup.362-613. These results show that residues
1-254 contain all the sequences essential for binding to PA63. The
fusion proteins had no toxicity in the absence of PA, proving that
their internalization absolutely requires interaction with PA.
Binding of Fusion Proteins and Deleted LF Proteins to PA
Binding of LF proteins to cell bound PA was determined by
competition with radiolabeled .sup.125 I-LF. Native LF was
radiolabeled (3.1.times.10.sup.6 cpm/.mu.g protein) using the
Bolton-Hunter reagent. Binding studies employed the L6 rat myoblast
cell line, which has approximately twice as many receptors as the
J774A.1 macrophage line (Singh et al., 1989). For convenience,
cells were chemically fixed by a gentle procedure that preserves
the binding activity of the receptor as well as the ability of the
cell-surface protease to cleave PA to produce receptor-bound PA63.
Assays were carried out in 24-well dishes using cells plated in
DMEM with 10% fetal bovine serum one day before the experiment.
Cell monolayers were washed twice with Hanks' balanced salt
solution (HBSS) containing 25 mM HEPES and were chemically fixed
for 30 min at 23.degree. in 10 mM N-hydroxysuccinimide and 30 mM
1-ethyl-3-[3-dimethyl[aminopropyl] carbodiimide, in buffer
containing 10 mM HEPES, 140 mM NaCl, 1 mM CaCl.sub.2, and 1 mM
MgCl.sub.2. Monolayers were washed with HBSS containing 25 mM HEPES
and the fixative was inactivated by incubating 30 min at 23.degree.
in DMEM (without serum) containing 25 mM HEPES. Native PA was added
at 1 .mu.g/ml in minimum essential medium containing Hanks' salts,
25 mM HEPES, 1% bovine serum albumin, and a total of 4.5 mM
NaHCO.sub.3. Cells were incubated overnight at room temperature to
allow binding and cleavage of PA. Cells were washed twice in HBSS
and mutant LF proteins (0-5000 ng/ml) along with 50 ng/ml .sup.125
I-LF was added to each well. Cells were further incubated for 5 h,
washed three times in HBSS, dissolved in 0.5 ml 1N NaOH, and
counted in a gamma counter (Beckman Gamma 9000).
Using this assay, the LF mutant proteins having amino-terminal
deletions were found incapable of binding to PA, thereby explaining
their lack of toxicity. Carboxyl-terminal deleted LF proteins did
bind to PA in a dose dependent manner, although they had slightly
lower affinity than LF. The proteins deleted in the repeat region
could not be tested for competitive binding because their
instability prevented purification of intact protein.
The EC.sub.50 for LF.sup.1-254.TR.PE.sup.362-613 binding was found
to be 220 ng/ml, which is similar to that of LF, 300 ng/ml.
Therefore the binding data correlate well with the toxicity of this
construct. In contrast, neither LF.sup.1-198.TR.PE.sup.362-613 nor
LF.sup.1-79.TR.PE.sup.362-613 bound to PA63 on cells, thereby
explaining their lack of toxicity.
EXAMPLE 3
Construction of Genes Encoding PA Fusion Proteins
The genes encoding PA (or PA truncated at the carboxyl terminus to
abrogate binding to the PA receptor) and an alternative targeting
moiety (a single-chain antibody, growth factor, or other cell
type-specific domain) are spliced using conventional molecular
biological techniques. The PA gene is readily available, and the
genes encoding alternative targeting domains are derived as
described below.
Single-chain antibodies (sFv)
See Example 4, below.
Growth Factors and Other Targeting Proteins
The nucleotide sequences of genes encoding a number of growth
factors and other proteins that are targeted to specific cell types
or classes are reported in freely accessible databases (e.g.,
GenBank), and in many cases the genes are available. In
circumstances where this is not the case, genes can be cloned from
genomic or cDNA libraries, using probes based on the known
nucleotide sequence of the gene that codes for the growth factor,
or derived from a partial amino acid sequence of the protein (see,
e.g. Sambrook, supra. ). Alternatively, genes encoding the growth
factor or other targeting moiety can be produced de novo from
chemically synthesized overlapping oligonucleotides, using the
preferred codon usage of the expression host. For example, the gene
for human epidermal growth factor urogastrone was synthesized from
the known amino acid sequence of human urogastrone using yeast
preferred codons. The cloned DNA, under control of the yeast GAPDH
promoter and yeast ADH-1 terminator, expresses a product having the
same properties as natural human urogastrone. The product of this
synthesized gene is nearly identical to that of the natural
urogastrone, the only difference being that the product of the
synthetic gene has a trptophan at amino acid 13, while the other
has a tyrosine (Urdea et al. Proc. Natl. Acad. Sci. USA
80:7461-7465, 1983).
Expression of PA Fusion Proteins
Once constructed, genes encoding PA-fusion proteins are expressed
in Bacillus anthracis, and recombinant proteins are purified by one
of the following methods: (i) size-based chromatographic
separation; (ii) affinity chromatography. In the case of PA-sFv
fusions, immobilized metal chelate affinity chromatography may be
the purification method of choice, because addition of a string of
six histidine residues at the carboxyl terminus of the sFv will
have no detrimental effect on binding to antigen. Additional
methods of expression of PA-fusion proteins utilize an in vitro
rabbit reticulocyte lysate-based coupled transcription/translation
system, which has been demonstrated to accurately refold chimeric
proteins consisting of an sFv fused to diphtheria toxin, or
Pseudomonas exotoxin A as demonstrated in Example 4.
Functional Testing of PA Fusion Proteins
After expression and purification, functionality of PA-fusion
proteins are tested by determining their ability to act in concert
with an LF-PE fusion protein to inhibit protein synthesis in an
appropriate cell line. Using a PA-anti human transferrin receptor
sFv fusion as a model, the following properties are examined: (i)
Cell type-specificity (protein synthesis should be inhibited in
cell lines which express the human transferrin receptor, but not in
those which do not); (ii) Independence of toxicity from PA receptor
binding (excess free PA should have no effect on toxicity of the
PA-sFv/LF-PE complex); (iii) Competitive inhibition by excess free
antibody (toxicity should be abrogated in the presence of excess
sFv, or the monoclonal antibody from which it was derived). For
example such tests are described in Examples 4 and 5. These studies
and other studies are used to confirm that PA has been successfully
re-routed to an alternative receptor to permit the use of the
present anthrax toxin-based cell type-specific cytotoxic agents for
the treatment of disease.
EXAMPLE 4
Generating Fusion Proteins with Single-Chain Antibodies
Reagents
Methionine-free rabbit reticulocyte lysate-based coupled
transcription/translation reagents, recombinant ribonuclease
inhibitor (rRNasin), and cartridges for the purification of plasmid
DNA were purchased from Promega (Madison, Wis.). Tissue culture
supplies were from GIBCO (Grand Island, N.Y.) and Biofluids
(Rockville, Md.). OKT9 monoclonal antibody was purchased from Ortho
Diagnostic Systems (Raritan, N.J.). PCR reagents were obtained from
by Perkin-Elmer Cetus Instruments (Norwalk, Conn.), and restriction
and nucleic acid modifying enzymes (including M-MLV reverse
transcriptase) were from GIBCO-BRL (Gaithersburg, Md.). A Geneclean
kit for the recovery of DNA from agarose gels was supplied by BIO
101 (La Jolla, Calif.). Hybridoma mRNA was isolated using a Fast
Trak mRNA isolation kit (Invitrogen, San Diego, Calif.). All
isotopes were purchased from Du Pont-New England Nuclear (Boston,
Mass.), except [Adenylate-.sup.32 P]NAD, which was supplied by ICN
Biomedicals (Costa Mesa, Calif.). Pseudomonas exotoxin A was
obtained from List Biologicals (Campbell, Calif.). Oligonucleotides
were synthesized on a dual column Milligen-Biosearch Cyclone Plus
DNA synthesizer (Burlington, Mass.), and purified using OPC
cartridges (Applied Biosystems, Foster City, Calif.). DNA templates
were sequenced using a Sequenase II kit (United States Biochemical
Corp., Cleveland, Ohio), and SDS-polyacrylamide gel electrophoresis
(PAGE) was performed using 10-20% gradient gels (Daiichi, Tokyo,
Japan). After electrophoresis, gels were fixed in 10% methanol/7%
acetic acid, and soaked in autoradiography enhancer (Amplify,
Amersham Arlington Heights, Ill.). After drying, autoradiography
was performed overnight using X-OMAT AR2 film (Eastman Kodak,
Rochester, N.Y.).
Plasmids
The vector pET-11d is available from Novagen, Inc., Madison, Wis.
Plasmids were maintained and propagated in E. coli strain XL1-Blue
(Stratagene, La Jolla, Calif.).
Cell Lines
K562, a human erythroleukemia-derived cell line [ATCC CCL 243]
known to express high levels of the human transferrin receptor at
the cell surface, was cultured in RPMI 1640 medium containing 24 mM
NaHCO.sub.3, 10% fetal calf serum, 2 mM glutamine, 1 mM sodium
pyruvate, 0.1 mM nonessential amino acids, and 10 .mu.g/ml
gentamycin. An African green monkey kidney line, Vero (ATCC CCL
81), was grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented as indicated above. The OKT9 hybridoma (ATCC CRL
8021), which produces a MoAb (IgG1) reactive to the human
transferrin receptor, was maintained in Iscove's modified
Dulbecco's medium containing 20% fetal calf serum, in addition to
the supplements described above. All cell lines were cultured at
37.degree. C. in a 5% CO.sub.2 humidified atmosphere.
Construction of sFv from Hybridomas
Antibody V.sub.L and V.sub.H genes were cloned using a modification
of a previously described technique (Larrick et al. Biotechniques
7:360, 1989; Orlandi et al. Proc. Natl. Acad. Sci. USA 86:3833,
1989; Chaudhary et al., 1990). Briefly, mRNA was isolated from
1.times.10.sup.8 antibody producing hybridoma cells, and
approximately 3 .mu.g was reverse transcribed with M-MLV reverse
transcriptase, using random hexanucleotides as primers. The
resulting cDNA was screened with two sets of PCR primer pairs
designed to ascertain from which Kabat gene family the heavy and
light chains were derived (Kabat et al. Sequences of proteins of
immunological interest. Fifth Edition. (Bethesda, Maryland: U.S.
Public Health Service, 1991). Having identified the most effective
primer pairs, cDNA's encoding V.sub.L and V.sub.H were spliced,
separated by a region encoding a 15 amino acid peptide linker,
using a previously described PCR technique known as gene splicing
by overlap extension (SOE) (Johnson & Bird Methods Enzymol.
203:88, 1991). The sFv gene was then cloned into pET-11d, in frame
and on the 5'-side of the PE40 gene, such that expression of the
construct should generate an sFv-PE40 fusion protein approximately
70 kDa in size.
Design of Primers for PCR Amplification of V Regions Genes
The first and third complementarity determining regions (CDRs) of
terminally rearranged immunoglobulin variable region genes are
flanked by conserved sequences (the first framework region, FR1 on
the 5' side of CDR1, and the fourth framework region, FR4, on the
3' side of CDR3).
Although murine variable region genes have been successfully
cloned, regardless of family, with just two pairs of highly
degenerate primers (one pair for V.sub.L and another for V.sub.H)
(Gussow et al. Cold Spring Harbor Symp. Quant. Biol. 54:265, 1989;
Orlandi et al., 1989; Chaudhary et al., 1990; Batra et al., 1991),
the method may not be effective in cases where the number of
mismatches between primers and the target sequence is extensive.
With this in mind, using the Kabat database of murine V gene
sequences the present invention provides a set of ten FR1-derived
primers (six for V.sub.L and four for V.sub.H), such that any of
the database sequences selected at random would have a maximum of
three mismatches with the most homologous primer. This set of
primers can be used effectively to clone V region genes from a
number of MoAb secreting cell lines.
Assembly of the OKT9 sFv Gene
mRNA isolated from the hybridoma secreting the OKT9 MoAb was
converted to cDNA as described previously (Larrick et al., 1989;
Orlandi et al., 1989; Chaudhary et al., 1990). Despite the fact
that CL-UNI is the partnering oligonucleotide in each case, a
product the required size (approximately 400 bp) is not produced by
V.sub.L primers IV/VI, IIa or IIb. This suggests that mismatches
between these primers and the target sequence were too extensive to
allow efficient amplification. A similar argument can be used to
explain the failure of V.sub.H primers I and III to produce the
required product. It is clear that primers V.sub.L -I/III and
V.sub.H -V are most effective at amplifying the OKT9 V.sub.L and
V.sub.H genes respectively. PCR amplified OKT9 V.sub.L and V.sub.H
genes were spliced together using the SOE technique, as previously
described (Johnson & Bird, 1991). A synthetic DNA sequence
encoding a 15 amino acid linker, was inserted between the variable
regions; this linker has been used very effectively in the
production of functional sFv (Huston et al., 1991; Johnson &
Bird, 1991), and appears to allow the variable chains to assume the
optimum orientation for antigen binding. Following splicing of V
region genes by the SOE procedure, the DNA fragment encoding the
OKT9 sFv was electrophoresed through a 1.5% agarose gel, purified
by the Geneclean technique, digested with the appropriate pair of
restriction enzymes, and cloned into the pET-11d expression vector
in frame and on the 5' side of the PE40 gene.
In Vitro Expression of sFv-PE40 Fusion Proteins
Plasmid templates were transcribed and translated using a rabbit
reticulocyte lysate-based transcription/translation system,
according to the instructions of the manufacturer, in 96-well
microtiter plate format L-[.sup.35 S]methionine-labeled proteins
(for analysis by SDS-PAGE) and unlabeled proteins (for enzymatic
analysis and bioassay), were produced in similar conditions, except
that the isotope was replaced with 20 .mu.M unlabeled L-methionine
in the latter case. Control lysate was produced by adding all
reagents except plasmid DNA. After translation, unlabeled samples
were dialysed overnight at 4.degree. C. against phosphate-buffered
saline (PBS), pH 7.4 in Spectra/Por 6 MWCO (molecular weight
cutoff) 50,000 tubing (Spectrum, Houston, Tex.).
Constructs incorporating the aberrant kappa transcript will contain
a translation termination codon in the V.sub.L chain as previously
described, and would therefore be expected to generate a
translation product approximately 12 kDa in size. On the other
hand, constructs which have incorporated the productive V.sub.L
gene contain no such termination codon, and a full-length fusion
protein (approximately 70 kDa in size) should be produced.
In vitro expression studies were used to determine the size of the
protein encoded by the OKT9 sFv-PE40 gene. The constructs tested in
this experiment clearly produce a protein of approximately 70 kDa,
indicating that the clones do not contain the aberrant V.sub.L
gene, and are devoid of frameshift mutations. Of several OKT9 sFv
constructs tested, none apparently incorporated the incorrect VL
gene. However, in the case of another sFv generated by this method
(1B7 sFv, derived from a MoAb which binds to pertussis toxin), the
majority of the clones tested produced a 12 kDa protein, and were
found to contain the aberrant transcript on DNA sequencing. It
should be noted that the 12 kDa fragment is frequently obscured in
10-20% gradient gels by unincorporated .sup.35 S-methionine which
co-migrates with the dye front.
Determination of Protein Concentration
The enzymatic activities of fusion proteins were compared with
those of known concentrations of PE in an ADP-ribosyl transferase
assay, allowing molarities to be determined (Johnson et al. J.
Biol. Chem. 263:1295-1399, 1988). Samples were adjusted to contain
equivalent concentrations of lysate, thus maintaining an identical
amount of substrate (elongation factor 2) in all cases.
Protein Synthesis Inhibition Assay for Functional sFv-PE40
Binding
Binding of the OKT9 sFv to the human transferrin receptor was
qualitatively determined by assessing the ability of the OKT9
sFv-PE40 fusion protein to inhibit protein synthesis in the K562
cell line. Pseudomonas exotoxin A is a bacterial protein which is
capable of inhibiting de novo protein synthesis in a variety of
eukaryotic cell types. The toxin binds to the cell surface, and
ultimately translocates to the cytosol where it enzymatically
inactivates elongation factor 2. PE40 is a mutant form of exotoxin
A which lacks a binding domain, but is enzymatically active, and
capable of translocation. Fusion proteins containing PE40 and an
alternative binding domain (for example, an sFv to a cell surface
receptor) will inhibit protein synthesis in an appropriate cell
line only if the sFv binds to a cell-surface antigen which
subsequently internalizes into an acidified endosome (Chaudhary et
al., 1989). The TfnR is such an antigen, so a qualitative
assessment of binding may be determined by measuring the ability of
the OKT9 sFv-PE40 fusion protein to inhibit protein synthesis in a
cell line like K562, which expresses the TfnR. Protein synthesis
inhibition assays were performed as described previously (Johnson
et al., 1988). Briefly, samples were serially diluted in ice cold
PBS, 0.2% BSA, and 11 .mu.l volumes were added to the appropriate
well of a 96-well microtiter plate (containing 10.sup.4 cells/100
.mu.l/well in leucine-free RPMI 1640). After carefully mixing the
contents of each well, the plate was incubated for the indicated
time at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere. Each
well was then pulsed with 20 .mu.l of L-[.sup.14 C(U)]leucine (0.1
.mu.Ci/20 .mu.l), incubated for 1 hour, and harvested onto glass
fiber filters using a PHD cell harvester (Cambridge Technology,
Cambridge, Mass.). Results are expressed as a percentage of the
isotope incorporation in cells treated with appropriate
concentrations of control dialyzed lysate.
The results of this assay, clearly indicate that OKT9 sFv-PE40 is
capable of inhibiting protein synthesis with an IC.sub.50 (the
concentration of a reagent which inhibits protein synthesis by 50%)
of approximately 2.times.10.sup.-9 M. The toxicity of the fusion
protein, but not of PE, was abrogated in the presence of excess
OKT9 MoAb (12 .mu.g/ml), indicating that binding is specific for
the TfnR. No toxicity was observed when K562 was substituted with
Vero (an African Green monkey cell line which expresses the simian
version of the transferrin receptor), indicating that the OKT9 sFv
retains the human receptor-specific antigen binding properties of
the parent antibody.
Having demonstrated binding of the OKT9 sFv to TfnR, its nucleotide
sequence was determined using dideoxynucleotide chain-terminating
methods, confirming extensive homology with the respective regions
of immunoglobulins of known sequence.
EXAMPLE 5
Characterization of Single-Chain Antibody (sFv)-Toxin Fusion
Proteins Produced in Vitro in Rabbit Reticulocyte Lysate
The present invention provides in vitro production of proteins
containing a toxin domain (derived from Diphtheria toxin (DT) or
PE) fused to a domain encoding a single-chain antibody directed
against the human transferrin receptor (TfnR). The expression of
this antigen on the cell surface is coordinately regulated with
cell growth; TfnR exhibits a limited pattern of expression in
normal tissue, but is widely distributed on carcinomas and sarcomas
(Gatter, et al. J. Clin. Pathol. 36:539-545, 1983), and may
therefore be a suitable target for immunotoxin-based therapeutic
strategies (Johnson, V. G. and Youle, R. J. "Intracellular
Trafficking of Proteins" Cambridge Univ. Press, Cambridge England,
Steer and Hover eds., pp. 183-225; Batra et al., 1991; Johnson et
al., 1988).
Proteins consisting of a fusion between an sFv directed against the
TfnR and either the carboxyl-terminus 40 kDa of PE, or the DT
mutant CRM 107 [S(525)F] were expressed in rabbit reticulocyte
lysates, and found to be specifically cytotoxic to K562, a cell
line known to express TfnR. In comparison, a chimeric protein
consisting of a fusion between a second DT mutant, DTM1 [S(508)F,
S(525)F] and the E6 sFv exhibited significantly lower cytotoxicity.
Legal restrictions imposed on manipulating toxin genes in vivo
previously prevented expression of potentially interesting
toxin-containing fusion proteins (Federal Register
51(88)(III):16961 and Appendix F:16971); the present invention
provides a novel procedure for in vitro gene construction and
expression which satisfies the regulatory requirements,
facilitating the first study of the potential of non-truncated DT
mutants in fusion protein ITs. The present data also demonstrates
that functional recombinant antibodies can be generated in
vitro.
Reagents
DT and PE were purchased from List Biologicals (Campbell, Calif.).
Nuclease treated, methionine-free rabbit reticulocyte lysate and
recombinant ribonuclease inhibitor (rRNasin) were obtained from
Promega (Madison, Wis.). Tissue culture supplies were from GIBCO
(Grand Island, N.Y.) and Biofluids (Rockville, Md.). Reagents for
PCR were provided by Perkin-Elmer Cetus (Norwalk, Conn.).
Restriction and nucleic acid modifying enzymes were from Stratagene
(La Jolla, Calif.), as was the mCAP kit used to produce capped mRNA
in vitro. Geneclean and RNaid kits (for the purification of DNA and
RNA respectively) were supplied by BIO 101 (La Jolla, Calif.).
L-[.sup.35 S]methionine, L-[.sup.14 C(U)]leucine and
5'-(alpha-thio)-[.sup.35 S]dATP were from New England Nuclear
(Boston, Mass.). [Adenylate-.sup.32 P]NAD was supplied by ICN
Biomedicals (Costa Mesa, Calif.).
Oligonucleotide Synthesis
Oligonucleotides were synthesized (0.2 .mu.M scale), using
cyanoethylphosphoramidites supplied by Milligen-Biosearch
(Burlington, Mass.) on a dual column Cyclone Plus DNA synthesizer.
Post-synthesis purification was achieved using OPC cartridges
(Applied Biosystems, Foster City, Calif.).
Plasmids
pET-11d was the generous gift of Dr. F. William Studier, Brookhaven
National Laboratory (Upton, N.Y.). pHB21-PE40, a derivative of
pET-11d containing the gene for PE40, was kindly supplied by Dr.
David FitzGerald (NIH, Bethesda, Md.). All plasmids were maintained
and propagated in E. coli strain XL1-Blue (Stratagene, La Jolla,
Calif.).
Cell Lines
Corynebacterium diphtheriae strain C7.sub.s (.beta.).sup.tox+ (ATCC
27012) was obtained from the ATCC (Rockville, Md.), and the strain
producing the binding-deficient DT mutant CRM 103 was the generous
gift of Dr. Neil Groman, University of Washington (Seattle, Wash.).
Both strains were propagated in LB broth. K562 (a human
erythroleukemia-derived cell line, ATCC CCL 243) was cultured in
RPMI 1640 medium containing 24 mM NaHCO.sub.3, 10% fetal calf
serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential
amino acids, and 10 .mu.g/ml gentamycin. Vero (an African green
monkey kidney line, ATCC CCL 81) was grown in Dulbecco's modified
Eagle's medium supplemented as described above. All eukaryotic
cells were cultured at 37.degree. C. in a 5% CO.sub.2 humidified
atmosphere.
Splicing Genes using PCR
Genes encoding antibody V.sub.L and V.sub.H were spliced, separated
by a region encoding a 15 amino acid peptide linker, using a
previously described PCR technique known as gene splicing by
overlap extension (SOE) (Horton et al. Gene 77:61-68, 1989; Horton
et al. Biotechniques 8:528-535, 1990). For studies requiring in
vitro expression of PCR products, tox gene-derived fragments were
linked to those encoding sFv using a similar method, without the
use of restriction enzymes.
Construction of Plasmids Encoding Toxin-sFv Fusion Proteins
The gene encoding PE40 was obtained as an insert in pET-11d, and
the sFv gene was cloned on the 5' side of this insert as indicated.
To clone the gene encoding the DT binding-site mutant DTM1
[S(508)F, S(525)F], genomic DNA was isolated from the C.
diphtheriae strain which produces CRM 103. DNA was extracted by a
modification of the cetyltrimethylammonium bromide extraction
procedure (Wilson, K. "Current Protocols in Molecular Biology"
Asubel et al. eds. John Wiley & Sons New York, 2.4.1-2.4.5,
1988) and subjected to 20 cycles of PCR amplification. Primers were
designed to: (i) amplify the 1605 bp region encoding CRM 103,
concomitantly mutating the codon at position 525 from TCT to TTT,
and (ii) incorporate restriction sites appropriate for cloning. The
mutations present in CRM 107 and CRM 103 were thus combined on a
single gene.
In Vitro Transcription of DNA Templates
For transcription, DNA templates required a T7 RNA polymerase
promoter immediately upstream of the gene of interest (Oakley, J.
L. and Coleman, J. E. Proc. Acad. Sci. U.S.A. 74:4266-4270, 1977).
Such a promoter was conveniently present in pET-11d (Studier et al.
Enzymol 185:60-89, 1990). In the case of PCR products, the upstream
primer (a 57-mer, T7-DT) was used to introduce all of the elements
necessary for in vitro transcription/translation. T7-DT includes a
consensus T7 RNA polymerase promoter, together with the first seven
codons of mature DT (Greenfield et al. Proc. Natl. Acad. Sci.
U.S.A. 80:6853-6857, 1983) immediately preceded by an ATG
translation initiation codon in the optimum Kozak context (Kozak,
M. J. Biol. Chem. 266:19867-19870, 1991). m.sup.7
G(5')ppp(5')G-capped RNA was produced by transcription from
linearized plasmids or PCR products using an mCAP kit, according to
the manufacturer's protocol. Prior to translation, RNA was purified
using an RNaid kit, recovered in nuclease free water, and analyzed
by formaldehyde gel electrophoresis.
In Vitro Expression of Fusion Proteins
L-[.sup.35 S]methionine-labelled proteins (for analysis by
SDS-PAGE) were produced from capped RNA in methionine-free,
nuclease treated rabbit reticulocyte lysate, according to the
supplier's instructions. Unlabeled proteins (for bioassay), were
produced in similar conditions, except that the isotope was
replaced with 20 .mu.M unlabeled L-methionine. Control lysate was
produced by adding all reagents except exogenous RNA. After
translation, samples were dialysed overnight at 4.degree. C.
against PBS, pH 7.4 in Spectra/Por 6 MWCO 50,000 tubing (Spectrum,
Houston, Tex.).
Prior to transcription, plasmids were linearized at the BglII site
and treated with proteinase K to destroy ribonucleases that may
contaminate the sample. After phenol/chloroform extraction and
ethanol precipitation, DNA was dissolved in nuclease free water to
a concentration of approximately 0.2 .mu.g/.mu.l. m.sup.7
G(5')ppp(5')G-capped RNA was synthesized by T7 RNA polymerase using
the conditions recommended by the manufacturer, and its integrity
was confirmed by formaldehyde gel electrophoresis. Capped RNA was
translated in a commercially available rabbit reticulocyte lysate,
according to the instructions of the manufacturer. It is clear from
the gel that the major band in each case has a molecular weight
corresponding to that of the protein of interest, and that
relatively large molecules (approximately 120 kDa in the case of
DTM1-E6 sFv-PE40) can be synthesized in the lysate using the
conditions described.
Immediately following translation, samples were extensively
dialyzed overnight at 4.degree. C. against PBS, pH 7.4. The
dialysis step was found to be essential, because non-dialyzed
rabbit reticulocyte lysate resulted in the incorporation of
significantly lower amounts of .sup.14 C-leucine upon assay by
protein synthesis inhibition in all cell lines tested. After
determining the concentration of the newly synthesized protein
using a standard assay for measuring ADP-ribosyltransferase
activity (Johnson et al., 1988), the cytotoxic activity of samples
was immediately determined.
ADP-ribosyl Transferase Assay
The enzymatic activity (and therefore molarity) of fusion proteins
was determined by comparison with DT or PE standard curves, as
described previously (Johnson et al., 1988). Appropriate volumes of
control lysate were added to each standard curve sample, in order
to control for the presence of significant levels of EF-2 in
reticulocyte lysate.
Other Methods
SDS-PAGE was performed as previously described (Laemmli, U. K.
Nature 227:680-685, 1970), using 10-20% gradient gels (Daiichi,
Tokyo, Japan). Once electrophoresis was complete, gels were fixed
for 15 minutes in 10% methanol, 7% acetic acid, and then soaked for
30 minutes in autoradiography enhancer (Amplify, Amersham Arlington
Heights, IL). After drying, autoradiography was performed overnight
using X-OMAT AR2 film (Eastman Kodak, Rochester, N.Y.), in the
absence of intensifying screens. Dideoxynucleotide
chain-termination sequencing of double-stranded DNA templates was
performed using a Sequenase II kit (United States Biochemical
Corp., Cleveland, Ohio), according to the manufacturer's
protocol.
Cytotoxicity of Toxin-sFv Fusion Proteins Expressed in Reticulocyte
Lysates
The cytotoxic activity of fusion proteins was determined by their
ability to inhibit protein synthesis in relevant cell lines (e.g.,
K562). Assays were performed as described previously (Johnson et
al., 1988). Briefly, samples were serially diluted in ice cold PBS,
0.2% BSA, and 11 .mu.l volumes were added to the appropriate well
of a 96-well microtiter plate (containing 10.sup.4 cells/well in
leucine-free RPMI 1640). After carefully mixing the contents of
each well, the plate was incubated for the indicated time at
37.degree. C. in a 5% CO.sub.2 humidified atmosphere. Each well was
then pulsed with 20 .mu.l of L-[.sup.14 C(U)]leucine (0.1 .mu.Ci/20
.mu.l), incubated for 1 hour, and harvested onto glass fiber
filters using a PHD cell harvester (Cambridge Technology,
Cambridge, Mass.). Results were expressed as a percentage of the
isotope incorporation in cells treated with appropriate
concentrations of control dialyzed lysate.
The results of the protein synthesis inhibition assay clearly
indicate that PE40-containing fusion proteins synthesized in
cell-free reticulocyte lysates are highly cytotoxic to this cell
line (IC.sub.50 1.times.10.sup.-10 M). In contrast, DTM1-E6 sFv was
at least ten-fold less toxic to K562 than the PE40-containing
fusion protein, despite the fact that it exhibited ADP-ribosyl
transferase activity indistinguishable from that of wt DT
synthesized from an equivalent amount of RNA in an identical
reticulocyte lysate mix. Since the decreased toxicity of DTM1-E6
sFv is clearly not due to a deficit in enzymatic activity, the
binding and/or translocation process is implicated. Possible
mechanisms by which the sFv-antigen interaction could be inhibited
include: (i) misfolding of the sFv domain or (ii) steric
interactions with other regions of the fusion protein preventing
close association of sFv with the TfnR. It is of interest that a
tripartite protein, DTM1-E6 sFv-PE40 was significantly cytotoxic to
K562 (IC.sub.50 around 1.times.10.sup.-10 M, similar to that of
PE40-E6 sFv), and the toxic effect was clearly mediated via the
TfnR, since this activity was blocked by addition of excess E6 Mab.
Although it is possible that the inclusion of the PE40 moiety at
the carboxyl end of the tripartite molecule results in a
significant conformational change in domains more proximal to the
amino terminus, it seems unlikely that the sFv binding domain of
DTM1-E6 is misfolded, or unavailable to interact with the TfnR.
Interactions of DTM1-E6 sFv with the cell surface could be measured
in a direct binding assay (Greenfield et al. Science 238:536-539,
1987), but these studies were not performed in the course of this
investigation. Nevertheless, it appears likely that the lack of
toxicity of the DTM1-E6 sFv fusion protein is due to a deficit in
its translocation function.
The expression system developed is rapid and easy, and facilitates
the manipulation of a number of samples at once. No complicated
protein purification or refolding procedures are required, and the
method can be used to express proteins which, due to restrictions
imposed on the manipulation of toxin-encoding genes, could not be
produced by more conventional methods. The technique is ideal for
ascertaining the suitability of new sFv for IT development; it is
theoretically possible to assemble the sFv-encoding gene (and that
encoding the IT itself) by splicing of PCR products derived
directly from the hybridoma, without the necessity for cloning.
This would facilitate the selection of the most promising candidate
molecule, prior to investing considerable effort and expense in
large scale protein production and purification. Toxins and
toxin-containing fusion proteins are proving to be powerful aids in
our understanding of receptor mediated endocytosis and
intracellular routing, and are providing valuable insight into
normal cell function (reviewed in ref. 2). The method described
simplifies the generation of such molecules, and facilitates their
production and use in laboratories in which the application of more
conventional expression methods would be impractical.
EXAMPLE 6
Cassette Mutagenesis to Produce PAHIV Mutants
Three pieces of DNA are joined together. Piece A has vector
sequences and encodes the "front half" (5' end of the gene) of PA
protein, B is short piece of DNA (referred to as a cassette) and
encodes a small middle piece of PA protein and piece C which
encodes the "back half" (3' end of the gene) of PA.
PA with alternate HIV-1 cleavage sites were created by a cassette
mutagenesis procedure. Eight deoxyoligonucleotides were synthesized
for construction of cassettes coding for specifically designed
amino acid sequences. All four cassettes were generated by
annealing two synthetic oligonucleotides (primers).
__________________________________________________________________________
Primer 1A CG CAA GTA TCA CAA AAT TAT CCG ATC GTG CAA AAC ATA CTG
CAG (SEQ ID NO: 18) Q V S Q N Y P I V Q N (SEQ ID NO: 19) Primer 1B
G TTC CTG CAG TAT GTT TTG CAC GAT CGG ATA ATT TTG TGA TAC (SEQ ID
NO: 20) Primer 2A CG AAC ACT GCC ACT ATC ATG ATG CAA CGT GGT AAT
TTT CTG CAG (SEQ ID NO: 21) N T A T I M M Q R G N (SEQ ID NO: 22)
Primer 2B G TCC CTG CAG AAA ATT ACC ACG TTG CAT CAT GAT AGT GGC AGT
(SEQ ID NO: 23) Primer 3A CG ACT GTC TCT TTT AAC TTC CCG CAA ATC
ACG CTT TGG CTG CAG (SEQ ID NO: 24) T V S F N F P Q I T L (SEQ ID
NO: 25) Primer 3B G TCC CTG CAG CCA AAG CGT GAT TTG CGG GAA GTT AAA
AGA GAC (SEQ ID NO: 26) Primer 4A CG GGC GGT TCT GCC TTT AAC TTC
CCG ATC GTC ATG GGA GGT CTG CAG (SEQ ID NO: 27) G G S A F N F P I V
M (SEQ ID NO: 28) Primer 4B G TCC CTG CAG ACC TCC CAT GAC GAT CGG
GAA GTT AAA GGC AGA ACC (SEQ ID NO:
__________________________________________________________________________
29)
The underlined portion of each protein sequence is recognized and
cleaved by the HIV-1 protease.
Primer pair 1 encodes a protein sequence which duplicates part of
the cleavage site found between the membrane associated protein and
the capsid protein.
Primer pair 2 encodes a protein sequence which duplicates part of
the cleavage site between the capsid and the nucleocapsid
protein.
Primer pair 3 encodes a protein sequence which duplicates part of
the cleavage site between the protease and the p6 protein. Like the
protease, p6 is a portion of the large protein produced by HIV.
Primer pair 4 encodes a protein sequence which should be cleaved by
the protease. It was created by examining several protein sequences
which are recognized by the HIV protease and using the common
residues from each sequence. Glycine residues were added to each
end to make the molecule more flexible.
The mutagenic cassettes were ligated with the BamHI/BstBI fragment
from plasmid pYS5 and the PpuMI-BamI-II fragment from plasmid pYS6.
Plasmids shown to have correct restriction maps were transformed
into the E. coli dam- dcm- strain GM2163 (available from New
England Bio-Labs, Beverly, Mass.). Unmethylated plasmid DNA was
purified from each mutant and used to transform B. anthracis. For
methods, see Klimpel, et al. Proc. Natl. Acad. Sci. 89:10277-10281
(1992). pYS5 and pYS6 construction are described in Singh, et al.
J. Bio. Chem. 264:19103-19107 (1989).
The nucleotide and amino acid sequence of the mature PA protein
after alteration with primer set 2 are shown below. Nucleotides
residues 482 to 523 were replaced with cassette 2 resulting in
replacement of amino acid residues 162-171 of PA with residues
NTATIMMQRGNFLQ (SEQ. ID NO: 22), PAHIV#2. The altered DNA sequence
and the new amino acid residues are underlined.
__________________________________________________________________________
Sequence Range: 1 to 2220 Nucleic acid sequences = (SEQ ID NO: 30);
Amino acid sequence = (SEQ ID NO: 31)
__________________________________________________________________________
60 * GAA GTT AAA CAG GAG AAC CGG TTA TTA AAT GAA TCA GAA TCA AGT
TCC CAG GGG TTA CTA CTT CAA TTT GTC CTC TTG GCC AAT AAT TTA CTT AGT
CTT AGT TCA AGG GTC CCC AAT GAT Glu Val Lys Gln Glu Asn Arg Leu Leu
Asn Glu Ser Glu Ser Ser Ser Gln Gly Leu Leu> 120 * GGA TAC TAT
TTT AGT GAT TTG AAT TTT CAA GCA CCC ATG GTG GTT ACC TCT TCT ACT ACA
CCT ATG ATA AAA TCA CTA AAC TTA AAA GTT CGT GGG TAC CAC CAA TGG AGA
AGA TGA TGT Gly Tyr Tyr Phe Ser Asp Leu Asn Phe Gln Ala Pro Met Val
Val Thr Ser Ser Thr Thr> 180 * GGG GAT TTA TCT ATT CCT AGT TCT
GAG TTA GAA AAT ATT CCA TCG GAA AAC CAA TAT TTT CCC CTA AAT AGA TAA
GGA TCA AGA CTC AAT CTT TTA TAA GGT AGC CTT TTG GTT ATA AAA Gly Asp
Leu Ser Ile Pro Ser Ser Glu Leu Glu Asn Ile Pro Ser Glu Asn Gln Tyr
Phe> 240 * CAA TCT GCT ATT TGG TCA GGA TTT ATC AAA GTT AAG AAG
AGT GAT GAA TAT ACA TTT GCT GTT AGA CGA TAA ACC AGT CCT AAA TAG TTT
CAA TTC TTC TCA CTA CTT ATA TGT AAA CGA Gln Ser Ala Ile Trp Ser Gly
Phe Ile Lys Val Lys Lys Ser Asp Glu Tyr Thr Phe Ala> 300 * ACT
TCC GCT GAT AAT CAT GTA ACA
ATG TGG GTA GAT GAC CAA GAA GTG ATT AAT AAA GCT TGA AGG CGA CTA TTA
GTA CAT TGT TAC ACC CAT CTA CTG GTT CTT CAC TAA TTA TTT CGA Thr Ser
Ala Asp Asn His Val Thr Met Trp Val Asp Asp Gln Glu Val Ile Asn Lys
Ala> 360 * TCT AAT TCT AAC AAA ATC AGA TTA GAA AAA GGA AGA TTA
TAT CAA ATA AAA ATT CAA TAT AGA TTA AGA TTG TTT TAG TCT AAT CTT TTT
CCT TCT AAT ATA GTT TAT TTT TAA GTT ATA Ser Asn Ser Asn Lys Ile Arg
Leu Glu Lys Gly Arg Leu Tyr Gln Ile Lys Ile Gln Tyr> 420 * CAA
CGA GAA AAT CCT ACT GAA AAA GGA TTG GAT TTC AAG TTG TAC TGG ACC GAT
TCT CAA GTT GCT CTT TTA GGA TGA CTT TTT CCT AAC CTA AAG TTC AAC ATG
ACC TGG CTA AGA GTT Gln Arg Glu Asn Pro Thr Glu Lys Gly Leu Asp Phe
Lys Leu Tyr Trp Thr Asp Ser Gln> 480 * AAT AAA AAA GAA GTG ATT
TCT AGT GAT AAC TTA CAA TTG CCA GAA TTA AAA CAA AAA TCT TTA TTT TTT
CTT CAC TAA AGA TCA CTA TTG AAT GTT AAC GGT CTT AAT TTT GTT TTT AGA
Asn Lys Lys Glu Val Ile Ser Ser Asp Asn Leu Gln Leu Pro Glu Leu Lys
Gln Lys Ser> 540 * ##STR1## AAC ACT GCC ACT ATC ATG ATG CAA CGT
GGT AAT TTT CTG CAG GGA CCT ACG GTT CCA AGC TTG TGA CGG
TGA TAG TAC TAC GTT GCA CCA TTA AAA GAC GTC CCT GGA TGC CAA GGT Ser
##STR2## Thr Ala Thr Ile Met Met Gln Arg Glu Asn Phe Leu Gln Gly
Pro Thr Val Pro> 600 * GAC CGT GAC AAT GAT GGA ATC CCT GAT TCA
TTA GAG GTA GAA GGA TAT ACG GTT GAT GTC CTC GCA CTG TTA CTA CCT TAG
GGA CTA AGT AAT CTC CAT CTT CCT ATA TGC CAA CTA CAG Asp Arg Asp Asn
Asp Gly Ile Pro Asp Ser Leu Glu Val Glu Gly Tyr Thr Val Asp Val>
660 * AAA AAT AAA AGA ACT TTT CTT TCA CCA TGG ATT TCT AAT ATT CAT
GAA AAG AAA GGA TTA TTT TTA TTT TCT TGA AAA GAA AGT GGT ACC TAA AGA
TTA TAA GTA CTT TTC TTT CCT AAT Lys Asn Lys Arg Thr Phe Leu Ser Pro
Trp Ile Ser Asn Ile His Glu Lys Lys Gly Leu> 720 * ACC AAA TAT
AAA TCA TCT CCT GAA AAA TGG AGC ACG GCT TCT GAT CCG TAC AGT GAT TTC
TGG TTT ATA TTT AGT AGA GGA CTT TTT ACC TCG TGC CGA AGA CTA GGC ATG
TCA CTA AAG Thr Lys Tyr Lys Ser Ser Pro Glu Lys Trp Ser Thr Ala Ser
Asp Pro Tyr Ser Asp Phe> 780 * GAA AAG GTT ACA GGA CGG ATT GAT
AAG AAT GTA TCA CCA GAG GCA AGA CAC CCC CTT GTG CTT TTC CAA TGT CCT
GCC TAA CTA TTC TTA CAT AGT GGT CTC CGT TCT GTG GGG GAA CAC
Glu Lys Val Thr Gly Arg Ile Asp Lys Asn Val Ser Pro Glu Ala Arg His
Pro Leu Val> 840 * GCA GCT TAT CCG ATT GTA CAT GTA GAT ATG GAG
AAT ATT ATT CTC TCA AAA AAT GAG GAT CGT CGA ATA GGC TAA CAT GTA CAT
CTA TAC CTC TTA TAA TAA GAG AGT TTT TTA CTC CTA Ala Ala Tyr Pro Ile
Val His Val Asp Met Glu Asn Ile Ile Leu Ser Lys Asn Glu Asp> 900
* CAA TCC ACA CAG AAT ACT GAT AGT GAA ACG AGA ACA ATA AGT AAA AAT
ACT TCT ACA AGT GTT
AGG TGT GTC TTA TGA CTA TCA CTT TGC TCT TGT TAT TCA TTT TTA TGA AGA
TGT TCA Gln Ser Thr Gln Asn Thr Asp Ser Glu Thr Arg Thr Ile Ser Lys
Asn Thr Ser Thr Ser> 960 * AGG ACA CAT ACT AGT GAA GTA CAT GGA
AAT GCA GAA GTG CAT GCG TCG TTC TTT GAT ATT TCC TGT GTA TGA TCA GTT
CAT GTA CCT TTA CGT CTT CAC GTA CGC AGC AAG AAA CTA TAA Arg Thr His
Thr Ser Glu Val His Gly Asn Ala Glu Val His Ala Ser Phe Phe Asp
Ile> 1020 * GGT GGG AGT GTA TCT GCA GGA TTT AGT AAT TCG AAT TCA
AGT ACG GTC GCA ATT GAT CAT CCA CCC TCA CAT AGA CGT CCT AAA TCA TTA
AGC TTA AGT TCA TGC CAG CGT TAA CTA GTA Gly Gly Ser Val Ser Ala Gly
Phe Ser Asn Ser Asn Ser Ser Thr Val Ala Ile Asp His> 1080 * TCA
CTA TCT CTA GCA GGG GAA AGA ACT TGG GCT GAA ACA ATG GGT TTA AAT ACC
GCT GAT AGT GAT AGA GAT CGT CCC CTT TCT TGA ACC CGA CTT TGT TAC CCA
AAT TTA TGG CGA CTA Ser Leu Ser Leu Ala Gly Glu Arg Thr Trp Ala Glu
Thr Met Gly Leu Asn Thr Ala Asp> 1140 * ACA GCA AGA TTA AAT GCC
AAT ATT AGA TAT GTA AAT ACT GGG ACG GCT CCA ATC TAC AAC TGT CGT TCT
AAT TTA CGG TTA TAA TCT ATA CAT TTA TGA CCC TGC CGA
GGT TAG ATG TTG Thr Ala Arg Leu Asn Ala Asn Ile Arg Tyr Val Asn Thr
Gly Thr Ala Pro Ile Tyr Asn> 1200 * GTG TTA CCA ACG ACT TCG TTA
GTG TTA GGA AAA AAT CAA ACA CTC GCG ACA ATT AAA GCT CAC AAT GGT TGC
TGA AGC AAT CAC AAT CCT TTT TTA GTT TGT GAG CGC TGT TAA TTT CGA Val
Leu Pro Thr Thr Ser Leu Val Leu Gly Lys Asn Gln Thr Leu Ala Thr Ile
Lys Ala> 1260 * AAG GAA AAC CAA TTA AGT CAA ATA CTT GCA CCT AAT
AAT TAT TAT CCT TCT AAA AAC TTG TTC CTT TTG GTT AAT TCA GTT TAT GAA
CGT GGA TTA TTA ATA ATA GGA AGA TTT TTG AAC Lys Glu Asn Gln Leu Ser
Gln Ile Leu Ala Pro Asn Asn Tyr Tyr Pro Ser Lys Asn Leu> 1320 *
GCG CCA ATC GCA TTA AAT GCA CAA GAC GAT TTC AGT TCT ACT CCA ATT ACA
ATG AAT TAC CGC GGT TAG CGT AAT TTA CGT GTT CTG CTA AAG TCA ACA TGA
GGT TAA TGT TAC TTA ATG Ala Pro Ile Ala Leu Asn Ala Gln Asp Asp Phe
Ser Ser Thr Pro Ile Thr Met Asn Tyr> 1440 * GGG AAT ATA GCA ACA
TAC AAT TTT GAA AAT GGA AGA GTG AGG GTG GAT ACA GGC TCG AAC CCC TTA
TAT GCT TGT ATG TTA AAA CTT TTA CCT TCT CAC TCC CAC CTA TGT CCG AGC
TTG Gly Asn Ile Ala Thr Tyr Asn Phe Glu Asn Gly Arg
Val Arg Val Asp Thr Gly Ser Asn 1500 * TGG AGT GAA GTG TTA CCG CAA
ATT CAA GAA ACA ACT GCA CGT ATC ATT TTT AAT GGA AAA ACC TCA CTT CAC
AAT GGC GTT TAA GTT CTT TGT TGA CGT GCA TAG TAA AAA TTA CCT TTT Trp
Ser Glu Val Leu Pro Gln Ile Gln Glu Thr Thr ALa Arg Ile Ile Phe Asn
Gly Lys 1560 * GAT TTA AAT CTG GTA GAA AGG CGG ATA GCG GCG GTT AAT
CCT AGT GAT CCA TTA GAA ACG CTA AAT TTA GAC CAT CTT TCC GCC TAT CGC
CGC CAA TTA GGA TCA CTA GGT AAT CTT TGC Asp Leu Asn Leu Val Glu Arg
Arg Ile Ala Ala Val Asn Pro Ser Asp Pro Leu Glu Thr 1620 * ACT AAA
CCG GAT ATG ACA TTA AAA GAA GCC CTT AAA ATA GCA TTT GGA TTT AAC GAA
CCG TGA TTT GGC CTA TAC TGT AAT TTT CTT CGG GAA TTT TAT CGT AAA CCT
AAA TTG CTT GGC Thr Lys Pro Asp Met Thr Leu Lys Glu Ala Leu Lys Ile
Ala Phe Gly Phe Asn Glu Pro 1680 * AAT GGA AAC TTA CAA TAT CAA GGG
AAA GAC ATA ACC GAA TTT GAT TTT AAT TTC GAT CAA TTA CCT TTG AAT GTT
ATA GTT CCC TTT CTG TAT TGG CTT AAA CTA AAA TTA AAG CTA GTT Asn Gly
Asn Leu Gln Tyr Gln Gly Lys Asp Ile Thr Glu Phe Asp Phe Asn Phe Asp
Gln 1740 * CAA ACA TCT CAA AAT ATC
AAG AAT CAG TTA GCG GAA TTA AAC GCA ACT AAC ATA TAT ACT GTT TGT AGA
GTT TTA TAG TTC TTA GTC AAT CGC CTT AAT TTG CGT TGA TTG TAT ATA TGA
Gln Thr Ser Gln Asn Ile Lys Asn Gln Leu Ala Glu Leu Asn Ala Thr Asn
Ile Tyr Thr 1800 * GTA TTA GAT AAA ATC AAA TTA AAT GCA AAA ATG AAT
ATT TTA ATA AGA GAT AAA CGT TTT CAT AAT CTA TTT TAG TTT AAT TTA CGT
TTT TAC TTA TAA AAT TAT TCT CTA TTT GCA AAA Val Leu Asp Lys Ile Lys
Leu Asn Ala Lys
Met Asn Ile Leu Ile Arg Asp Lys Arg Phe 1860 * CAT TAT GAT AGA AAT
AAC ATA GCA GTT GGG GCG GAT GAG TCA GTA GTT AAG GAG GCT CAT GTA ATA
CTA TCT TTA TTG TAT CGT CAA CCC CGC CTA CTC AGT CAT CAA TTC CTC CGA
GTA His Tyr Asp Arg Asn Asn Ile Ala Val Gly Ala Asp Glu Ser Val Val
Lys Glu Ala His 1920 * AGA GAA GTA ATT AAT TCG TCA ACA GAG GGA TTA
TTG TTA AAT ATT GAT AAG GAT ATA AGA TCT CTT CAT TAA TTA AGC AGT TGT
CTC CCT AAT AAC AAT TTA TAA CTA TTC CTA TAT TCT Arg Glu Val Ile Asn
Ser Ser Thr Glu Gly Leu Leu Leu Asn Ile Asp Lys Asp Ile Arg 1980 *
AAA ATA TTA TCA GGT TAT ATT GTA GAA ATT GAA GAT ACT GAA GGG CTT AAA
GAA GTT ATA TTT TAT AAT AGT CCA ATA TAA CAT CTT TAA CTT CTA TGA CTR
CCC GAA TTT CTT CAA TAT Lys Ile Leu Ser Gly Tyr Ile Val Glu Ile Glu
Asp Thr Glu Gly Leu Lys Glu Val Ile 2040 * AAT GAC AGA TAT GAT ATG
TTG AAT ATT TCT AGT TTA CGG CAA GAT GGA AAA ACA TTT ATA TTA CTG TCT
ATA CTA TAC AAC TTA TAA AGA TCA AAT GCC CTT CTA CCT TTT TGT AAA TAT
Asn Asp Arg Tyr Asp Met Leu Asn IIe Ser Ser Leu Arg Gln Asp Gly Lys
Thr Phe Ile 2100 * GAT TTT AAA AAA
TAT AAT GAT AAA TTA CCG TTA TAT ATA AGT AAT CCC AAT TAT AAG GTA CTA
AAA TTT TTT ATA TTA CTA TTT AAT GGC AAT ATA TAT TCA TTA GGG TTA ATA
TTC CAT Asp Phe Lys Lys Tyr Asn Asp Lys Leu Pro Leu Tyr Ile Ser Asn
Pro Asn Tyr Lys Val 2160 * AAT GTA TAT GCT GTT ACT AAA GAA AAC ACT
ATT ATT AAT CCT AGT GAG AAT GGG GAT ACT TTA CAT ATA CGA CAA TGA TTT
CTT TTG TGA TAA TAA TTA GGA TCA CTC TTA CCC CTA TGA Asn Val Tyr Ala
Val Thr Lys Glu Asn Thr Ile Ile Asn Pro Ser Glu Asn Gly Asp Thr
2220 * AGT ACC AAC GGG ATC AAG AAA ATT TTA ATC TTT TCT AAA AAA GGC
TAT GAG ATA GGA TAA TCA TGG TTG CCC TAG TTC TTT TAA AAT TAG AAA AGA
TTT TTT CCG ATA CTC TAT CCT ATT Ser Thr Asn Gly Ile Lys Lys Ile Leu
Ile Phe Ser Lys Lys Gly Tyr Glu Ile Gly***
__________________________________________________________________________
The above procedure was followed for PAHIV#1, 3 and 4.
EXAMPLE 7
Cleavage of Mutant PAHIV Proteins in vitro
The mutated proteins were treated with purified HIV-1 protease and
evaluated for their degree of cleavage with respect to time. The
purified protease was obtained from the NIH AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, Bethesda, Md.
Alternatively, the protease can be purified following the method of
Louis, et al., Euro. J. Biochem., 199:361 (1991).
Extended incubation (12 hours) of PA or the mutated PA proteins
with the purified HIV-1 protease resulted in the appearance of two
additional protein fragments that were not anticipated. These two
fragments are approximately 53 kilodaltons and 30 kilodaltons in
size. This may represent cleavage of PA and mutant PA proteins at a
site recognized by the HIV-1 protease between PA residues y.sup.259
and p.sup.260. The residues around this cleavage site, .sup.256
VAAYPIVHV.sup.264 (SEQ. ID NO: 35), have not previously been
identified as a potential HIV-1 protease cleavage site.
Incubation of RAW 264.7 cells (ATCC No. TIB 71) with lethal factor
(LF) and HIV-1 protease-cleaved PAHIV#1 or PAHIV#4 caused cell
death, demonstrating that the mutated PA proteins are capable of
binding to LF and thus the toxic LF/PE fusion proteins. PAHIV,
PAHIV#2 and PAHIV#3 have not yet been tested.
EXAMPLE 8
Evaluation of Cytotoxic Agents in Cell Cultures
The ability of the PA constructs containing the HIV-1-protease
cleavage site to promote killing of HIV-1 infected cells is being
evaluated in COS-1 cells (ATCC No. CRL 1650) transfected with the
vector HIV-gpt. When COS cells are transfected with this plasmid
vector they express all the genes for the production of HIV-1 virus
particles except the envelope protein, gp160 (Page, K. A., et al.,
1990. J. Virol. 64:5270-5276). Without the envelope protein the
particles are not infectious. These cells express the HIV-1
proteases and properly cleave the viral protein gp55 to gp24 (Page,
K. A., et al., 1990. J. Virol. 64:5270-5276). These properties make
the transfected cells an excellent model system in which to
evaluate the ability of protein constructs of the invention to
eliminate HIV-1 infected cells from culture.
The COS-1 cells were transfected with the plasmid vector and the
resulting cultures are being selected for stable transfectents. The
mutated PA proteins (PAHIV#1, PAHIV#2, PAHIV#3 and PAHIV#4) are
added to the culture media of growing HIV-gpt transfected COS-1
cells in the presence of the lethal factor fusion protein FP53
(Arora, N. et al. J. Biol. Chem. 267:15542 (1992)). Only cells
which properly cleave the mutated PA proteins are able to bind the
toxin LF fusion protein. The cultures are evaluated for protein
expression (an indirect measure of viability) after 36 hours
(Arora, N. and S. H. Leppla. 1992. J. Biol. Chem. 268:3334).
EXAMPLE 9
Treatment of an HIV-1 infected patient
A human patient who is infected with HIV-1 is selected for
treatment. Although infected, this particular patient is
asymptomatic. The patient weighs 70 kilograms. A dose of 10
micrograms per kilogram or 700 micrograms of a PAHIV in normal
saline is prepared. This dosage is injected into the patient
intravenously as a bolus. The dose is repeated weekly for a total
of 4 to 6 dosages. The patient is evaluated regularly, such as
weekly, in terms of his symptoms, physical exam and laboratory
analysis according to the clinician's judgment. Tests of particular
interest include the patient's complete blood count and examination
for the presence of HIV infection. The treatment regimen can be
repeated with or without alterations at the discretion of the
clinician.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
can be used in the practice or testing of the present invention,
the preferred methods and materials are now described. All
publications and patent documents referenced in this application
are incorporated herein by reference.
It is understood that the examples and embodiments described herein
are for illustrative purposes only and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 35 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 3291 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 580..2907 (D) OTHER INFORMATION:
/product="Lethal Factor" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AAATTAGGATTTCGGTTATGTTTAGTATTTTTTTAAAATAATAGTATTAAATAGTGGAAT60
GCAAATGATAAATGGGCTTTAAACAAAACTAATGAAATAATCTACAAATGGAATTTCTCC120
AGTTTTAGATTAAACCATACCAAAAAAATCACACTGTCAAGAAAAATGATAGAATCCCTA180
CACTAATTAACATAACCAAATTGGTAGTTATAGGTAGAAACTTATTTATTTCTATAATAC240
CATGCAAAAAAGTAAATATTCTGTTCCATACTATTTTAGTAAATTATTTAGCAAGTAAAT300
TTTGGTGTATAAACAAAGTTTATCTTAATATAAAAAATTACTTTACTTTTATACAGATTA360
AAATGAAAAATTTTTTATGACAAGAAATATTGCCTTTAATTTATGAGGAAATAAGTAAAA420
TTTTCTACATACTTTATTTTATTGTTGAAATGTTCACTTATAAAAAAGGAGAGATTAAAT480
ATGAATATAAAAAAAGAATTTATAAAAGTAATTAGTATGTCATGTTTAGTAACAGCAATT540
ACTTTGAGTGGTCCCGTCTTTATCCCCCTTGTACAGGGGGCGGGCGGTCATGGT594
AlaGlyGlyHisGly 15
GATGTAGGTATGCACGTAAAAGAGAAAGAGAAAAATAAAGATGAGAAT642
AspValGlyMetHisValLysGluLysGluLysAsnLysAspGluAsn 101520
AAGAGAAAAGATGAAGAACGAAATAAAACACAGGAAGAGCATTTAAAG690
LysArgLysAspGluGluArgAsnLysThrGlnGluGluHisLeuLys 253035
GAAATCATGAAACACATTGTAAAAATAGAAGTAAAAGGGGAGGAAGCT738
GluIleMetLysHisIleValLysIleGluValLysGlyGluGluAla 404550
GTTAAAAAAGAGGCAGCAGAAAAGCTACTTGAGAAAGTACCATCTGAT786
ValLysLysGluAlaAlaGluLysLeuLeuGluLysValProSerAsp 556065
GTTTTAGAGATGTATAAAGCAATTGGAGGAAAGATATATATTGTGGAT834
ValLeuGluMetTyrLysAlaIleGlyGlyLysIleTyrIleValAsp 70758085
GGTGATATTACAAAACATATATCTTTAGAAGCATTATCTGAAGATAAG882
GlyAspIleThrLysHisIleSerLeuGluAlaLeuSerGluAspLys 9095100
AAAAAAATAAAAGACATTTATGGGAAAGATGCTTTATTACATGAACAT930
LysLysIleLysAspIleTyrGlyLysAspAlaLeuLeuHisGluHis 105110115
TATGTATATGCAAAAGAAGGATATGAACCCGTACTTGTAATCCAATCT978
TyrValTyrAlaLysGluGlyTyrGluProValLeuValIleGlnSer 120125130
TCGGAAGATTATGTAGAAAATACTGAAAAGGCACTGAACGTTTATTAT1026
SerGluAspTyrValGluAsnThrGluLysAlaLeuAsnValTyrTyr 135140145
GAAATAGGTAAGATATTATCAAGGGATATTTTAAGTAAAATTAATCAA1074
GluIleGlyLysIleLeuSerArgAspIleLeuSerLysIleAsnGln 150155160165
CCATATCAGAAATTTTTAGATGTATTAAATACCATTAAAAATGCATCT1122
ProTyrGlnLysPheLeuAspValLeuAsnThrIleLysAsnAlaSer 170175180
GATTCAGATGGACAAGATCTTTTATTTACTAATCAGCTTAAGGAACAT1170
AspSerAspGlyGlnAspLeuLeuPheThrAsnGlnLeuLysGluHis 185190195
CCCACAGACTTTTCTGTAGAATTCTTGGAACAAAATAGCAATGAGGTA1218
ProThrAspPheSerValGluPheLeuGluGlnAsnSerAsnGluVal 200205210
CAAGAAGTATTTGCGAAAGCTTTTGCATATTATATCGAGCCACAGCAT1266
GlnGluValPheAlaLysAlaPheAlaTyrTyrIleGluProGlnHis 215220225
CGTGATGTTTTACAGCTTTATGCACCGGAAGCTTTTAATTACATGGAT1314
ArgAspValLeuGlnLeuTyrAlaProGluAlaPheAsnTyrMetAsp 230235240245
AAATTTAACGAACAAGAAATAAATCTATCCTTGGAAGAACTTAAAGAT1362
LysPheAsnGluGlnGluIleAsnLeuSerLeuGluGluLeuLysAsp 250255260
CAACGGATGCTGTCAAGATATGAAAAATGGGAAAAGATAAAACAGCAC1410
GlnArgMetLeuSerArgTyrGluLysTrpGluLysIleLysGlnHis 265270275
TATCAACACTGGAGCGATTCTTTATCTGAAGAAGGAAGAGGACTTTTA1458
TyrGlnHisTrpSerAspSerLeuSerGluGluGlyArgGlyLeuLeu 280285290
AAAAAGCTGCAGATTCCTATTGAGCCAAAGAAAGATGACATAATTCAT1506
LysLysLeuGlnIleProIleGluProLysLysAspAspIleIleHis 295300305
TCTTTATCTCAAGAAGAAAAAGAGCTTCTAAAAAGAATACAAATTGAT1554
SerLeuSerGlnGluGluLysGluLeuLeuLysArgIleGlnIleAsp 310315320325
AGTAGTGATTTTTTATCTACTGAGGAAAAAGAGTTTTTAAAAAAGCTA1602
SerSerAspPheLeuSerThrGluGluLysGluPheLeuLysLysLeu 330335340
CAAATTGATATTCGTGATTCTTTATCTGAAGAAGAAAAAGAGCTTTTA1650
GlnIleAspIleArgAspSerLeuSerGluGluGluLysGluLeuLeu 345350355
AATAGAATACAGGTGGATAGTAGTAATCCTTTATCTGAAAAAGAAAAA1698
AsnArgIleGlnValAspSerSerAsnProLeuSerGluLysGluLys 360365370
GAGTTTTTAAAAAAGCTGAAACTTGATATTCAACCATATGATATTAAT1746
GluPheLeuLysLysLeuLysLeuAspIleGlnProTyrAspIleAsn 375380385
CAAAGGTTGCAAGATACAGGAGGGTTAATTGATAGTCCGTCAATTAAT1794
GlnArgLeuGlnAspThrGlyGlyLeuIleAspSerProSerIleAsn 390395400405
CTTGATGTAAGAAAGCAGTATAAAAGGGATATTCAAAATATTGATGCT1842
LeuAspValArgLysGlnTyrLysArgAspIleGlnAsnIleAspAla 410415420
TTATTACATCAATCCATTGGAAGTACCTTGTACAATAAAATTTATTTG1890
LeuLeuHisGlnSerIleGlySerThrLeuTyrAsnLysIleTyrLeu 425430435
TATGAAAATATGAATATCAATAACCTTACAGCAACCCTAGGTGCGGAT1938
TyrGluAsnMetAsnIleAsnAsnLeuThrAlaThrLeuGlyAlaAsp 440445450
TTAGTTGATTCCACTGATAATACTAAAATTAATAGAGGTATTTTCAAT1986
LeuValAspSerThrAspAsnThrLysIleAsnArgGlyIlePheAsn 455460465
GAATTCAAAAAAAATTTCAAATATAGTATTTCTAGTAACTATATGATT2034
GluPheLysLysAsnPheLysTyrSerIleSerSerAsnTyrMetIle 470475480485
GTTGATATAAATGAAAGGCCTGCATTAGATAATGAGCGTTTGAAATGG2082
ValAspIleAsnGluArgProAlaLeuAspAsnGluArgLeuLysTrp 490495500
AGAATCCAATTATCACCAGATACTCGAGCAGGATATTTAGAAAATGGA2130
ArgIleGlnLeuSerProAspThrArgAlaGlyTyrLeuGluAsnGly 505510515
AAGCTTATATTACAAAGAAACATCGGTCTGGAAATAAAGGATGTACAA2178
LysLeuIleLeuGlnArgAsnIleGlyLeuGluIleLysAspValGln 520525530
ATAATTAAGCAATCCGAAAAAGAATATATAAGGATTGATGCGAAAGTA2226
IleIleLysGlnSerGluLysGluTyrIleArgIleAspAlaLysVal 535540545
GTGCCAAAGAGTAAAATAGATACAAAAATTCAAGAAGCACAGTTAAAT2274
ValProLysSerLysIleAspThrLysIleGlnGluAlaGlnLeuAsn 550555560565
ATAAATCAGGAATGGAATAAAGCATTAGGGTTACCAAAATATACAAAG2322
IleAsnGlnGluTrpAsnLysAlaLeuGlyLeuProLysTyrThrLys 570575580
CTTATTACATTCAACGTGCATAATAGATATGCATCCAATATTGTAGAA2370
LeuIleThrPheAsnValHisAsnArgTyrAlaSerAsnIleValGlu 585590595
AGTGCTTATTTAATATTGAATGAATGGAAAAATAATATTCAAAGTGAT2418
SerAlaTyrLeuIleLeuAsnGluTrpLysAsnAsnIleGlnSerAsp 600605610
CTTATAAAAAAGGTAACAAATTACTTAGTTGATGGTAATGGAAGATTT2466
LeuIleLysLysValThrAsnTyrLeuValAspGlyAsnGlyArgPhe 615620625
GTTTTTACCGATATTACTCTCCCTAATATAGCTGAACAATATACACAT2514
ValPheThrAspIleThrLeuProAsnIleAlaGluGlnTyrThrHis 630635640645
CAAGATGAGATATATGAGCAAGTTCATTCAAAAGGGTTATATGTTCCA2562
GlnAspGluIleTyrGluGlnValHisSerLysGlyLeuTyrValPro 650655660
GAATCCCGTTCTATATTACTCCATGGACCTTCAAAAGGTGTAGAATTA2610
GluSerArgSerIleLeuLeuHisGlyProSerLysGlyValGluLeu 665670675
AGGAATGATAGTGAGGGTTTTATACACGAATTTGGACATGCTGTGGAT2658
ArgAsnAspSerGluGlyPheIleHisGluPheGlyHisAlaValAsp 680685690
GATTATGCTGGATATCTATTAGATAAGAACCAATCTGATTTAGTTACA2706
AspTyrAlaGlyTyrLeuLeuAspLysAsnGlnSerAspLeuValThr 695700705
AATTCTAAAAAATTCATTGATATTTTTAAGGAAGAAGGGAGTAATTTA2754
AsnSerLysLysPheIleAspIlePheLysGluGluGlySerAsnLeu 710715720725
ACTTCGTATGGGAGAACAAATGAAGCGGAATTTTTTGCAGAAGCCTTT2802
ThrSerTyrGlyArgThrAsnGluAlaGluPhePheAlaGluAlaPhe 730735740
AGGTTAATGCATTCTACGGACCATGCTGAACGTTTAAAAGTTCAAAAA2850
ArgLeuMetHisSerThrAspHisAlaGluArgLeuLysValGlnLys 745750755
AATGCTCCGAAAACTTTCCAATTTATTAACGATCAGATTAAGTTCATT2898
AsnAlaProLysThrPheGlnPheIleAsnAspGlnIleLysPheIle 760765770
ATTAACTCATAAGTAATGTATTAAAAATTTTCAAATGGATTTAATAATA2947 IleAsnSer 775
ATAATAATAATAATAATAACGGGACCAGCCATTATGAAGCAACTAATTCTAGACTTGATA3007
GTAATTCTTGGGAAGCACCAGATAGTGTAAAAGGTGGCATTGCCAGAATGATATTTTATG3067
TGTTCGTTAGATATGAAGGCAAAAACAATGATCCTGACCTAGAACTTAATGATAATGTTA3127
TTAATAATTTAATGCCTTTTATAGGAATATTAGTAAAAGTGCCGAAAAGATCCTGTTGCA3187
AAGCTTTTAAAGAACATATTATTCTATCAAGTGGCTGTATATTTTGTGTAATTTTCAATA3247
AATTTTGTAATTAAGCATACGTCAAAAAACCGAAATCTGAGCTC3291 (2) INFORMATION
FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 776
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
AlaGlyGlyHisGlyAspValGlyMetHisValLysGluLysGluLys 151015
AsnLysAspGluAsnLysArgLysAspGluGluArgAsnLysThrGln 202530
GluGluHisLeuLysGluIleMetLysHisIleValLysIleGluVal 354045
LysGlyGluGluAlaValLysLysGluAlaAlaGluLysLeuLeuGlu 505560
LysValProSerAspValLeuGluMetTyrLysAlaIleGlyGlyLys 65707580
IleTyrIleValAspGlyAspIleThrLysHisIleSerLeuGluAla 859095
LeuSerGluAspLysLysLysIleLysAspIleTyrGlyLysAspAla 100105110
LeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyrGluProVal 115120125
LeuValIleGlnSerSerGluAspTyrValGluAsnThrGluLysAla 130135140
LeuAsnValTyrTyrGluIleGlyLysIleLeuSerArgAspIleLeu 145150155160
SerLysIleAsnGlnProTyrGlnLysPheLeuAspValLeuAsnThr 165170175
IleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeuPheThrAsn 180185190
GlnLeuLysGluHisProThrAspPheSerValGluPheLeuGluGln 195200205
AsnSerAsnGluValGlnGluValPheAlaLysAlaPheAlaTyrTyr 210215220
IleGluProGlnHisArgAspValLeuGlnLeuTyrAlaProGluAla 225230235240
PheAsnTyrMetAspLysPheAsnGluGlnGluIleAsnLeuSerLeu 245250255
GluGluLeuLysAspGlnArgMetLeuSerArgTyrGluLysTrpGlu 260265270
LysIleLysGlnHisTyrGlnHisTrpSerAspSerLeuSerGluGlu 275280285
GlyArgGlyLeuLeuLysLysLeuGlnIleProIleGluProLysLys 290295300
AspAspIleIleHisSerLeuSerGlnGluGluLysGluLeuLeuLys 305310315320
ArgIleGlnIleAspSerSerAspPheLeuSerThrGluGluLysGlu 325330335
PheLeuLysLysLeuGlnIleAspIleArgAspSerLeuSerGluGlu 340345350
GluLysGluLeuLeuAsnArgIleGlnValAspSerSerAsnProLeu 355360365
SerGluLysGluLysGluPheLeuLysLysLeuLysLeuAspIleGln 370375380
ProTyrAspIleAsnGlnArgLeuGlnAspThrGlyGlyLeuIleAsp 385390395400
SerProSerIleAsnLeuAspValArgLysGlnTyrLysArgAspIle 405410415
GlnAsnIleAspAlaLeuLeuHisGlnSerIleGlySerThrLeuTyr 420425430
AsnLysIleTyrLeuTyrGluAsnMetAsnIleAsnAsnLeuThrAla 435440445
ThrLeuGlyAlaAspLeuValAspSerThrAspAsnThrLysIleAsn 450455460
ArgGlyIlePheAsnGluPheLysLysAsnPheLysTyrSerIleSer
465470475480 SerAsnTyrMetIleValAspIleAsnGluArgProAlaLeuAspAsn
485490495 GluArgLeuLysTrpArgIleGlnLeuSerProAspThrArgAlaGly
500505510 TyrLeuGluAsnGlyLysLeuIleLeuGlnArgAsnIleGlyLeuGlu
515520525 IleLysAspValGlnIleIleLysGlnSerGluLysGluTyrIleArg
530535540 IleAspAlaLysValValProLysSerLysIleAspThrLysIleGln
545550555560 GluAlaGlnLeuAsnIleAsnGlnGluTrpAsnLysAlaLeuGlyLeu
565570575 ProLysTyrThrLysLeuIleThrPheAsnValHisAsnArgTyrAla
580585590 SerAsnIleValGluSerAlaTyrLeuIleLeuAsnGluTrpLysAsn
595600605 AsnIleGlnSerAspLeuIleLysLysValThrAsnTyrLeuValAsp
610615620 GlyAsnGlyArgPheValPheThrAspIleThrLeuProAsnIleAla
625630635640 GluGlnTyrThrHisGlnAspGluIleTyrGluGlnValHisSerLys
645650655 GlyLeuTyrValProGluSerArgSerIleLeuLeuHisGlyProSer
660665670 LysGlyValGluLeuArgAsnAspSerGluGlyPheIleHisGluPhe
675680685 GlyHisAlaValAspAspTyrAlaGlyTyrLeuLeuAspLysAsnGln
690695700 SerAspLeuValThrAsnSerLysLysPheIleAspIlePheLysGlu
705710715720 GluGlySerAsnLeuThrSerTyrGlyArgThrAsnGluAlaGluPhe
725730735 PheAlaGluAlaPheArgLeuMetHisSerThrAspHisAlaGluArg
740745750 LeuLysValGlnLysAsnAlaProLysThrPheGlnPheIleAsnAsp
755760765 GlnIleLysPheIleIleAsnSer 770775 (2) INFORMATION FOR SEQ
ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4235 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1891..4095
(D) OTHER INFORMATION: /product="Protective Antigen" (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:3:
AAGCTTCTGTCATTCGTAAATTTCAAATAGAACGTAAATTTAGACTTCTCATCATTAAAA60
ATGAAAAATCTTATCTTTTTGATTCTATTGTATATTTTTATTAAGGTGTTTAATAGTTAG120
AAAAGACAGTTGATGCTATTACTCCAGATAAAATATAGCTAACCATAAATTTATTAAAGA180
AACCTTGTTGTTCTAAATAATGATTTTGTGGATTCCGGAATAGATACTGGTGAGTTAGCT240
CTAATTTTATAGTGATTTAACTAACAATTTATAAAGCAGCATAATTCAAATTTTTTAATT300
GATTTTTCCTGAAGCATAGTATAAAAGAGTCAAGGTCTTCTAGACTTGACTCTTGGAATC360
ATTAGGAATTAACAATATATATAATGCGCTAGACAGAATCAAATTAAATGCAAAAATGAA420
TATTTTAGTAAGAGATCCATATCATTATGATAATAACGGTAATATTGTAGGGGTTGATGA480
TTCATATTTAAAAAACGCATATAAGCAAATACTTAATTGGTCAAGCGATGGAGTTTCTTT540
AAATCTAGATGAAGATGTAAATCAAGCACTATCTGGATATATGCTTCAAATAAAAAAACC600
TTCAAACCACCTAACAAACAGCCCAGTTACAATTACATTAGCAGGCAAGGACAGTGGTGT660
TGGAGAATTGTATAGAGTATTATCAGATGGAGCAGGATTCCTGGATTTCAATAAGTTTGA720
TGAAAATTGGCGATCATTAGTAGATCCTGGTGATGATGTTTATGTGTATGCTGTTACTAA780
AGAAGATTTTAATGCAGTTACTCGAGATGAAAATGGTAATATAGCGAATAAATTAAAAAA840
CACCTTAGTTTTATCGGGTAAAATAAAAGAAATAAACATAAAAACTACAAATATTAATAT900
ATTTGTAGTTTTTATGTTTATTATATACCTCCTATTTTATATTATTAGTAGCACAGTTTT960
TGCAAATCATGTAATTGTATACTTATCTATGTAGAGGTATCACAACTTATGAATAGTGTA1020
TTTTATTGAACGTTGGTTAGCTTGGACAGTTGTATGGATATGCATACTTTATAACGTATA1080
AAATTTCACGCACCACAATAAAACTAATTTAACAAAAACAAAAACACACCTAAGATCATT1140
CAGTTCTTTTAATAAGGAGCTGCCCACCAAGCTAAACCTAAATAATCTTTGTTTCACATA1200
AGGTTTTTTTCTAAATATACAGTGTAAGTTATTGTGAATTTAACCAGTATATATTAAAAA1260
TGTTTTATGTTAACAAATTAAATTGTAAAACCCCTCTTAAGCATAGTTAAGAGGGGTAGG1320
TTTTAAATTTTTTGTTGAAATTAGAAAAAATAATAAAAAAACAAACCTATTTTCTTTCAG1380
GTTGTTTTTGGGTTACAAAACAAAAAGAAAACATGTTTCAAGGTACAATAATTATGGTTC1440
TTTAGCTTTCTGTAAAACAGCCTTAATAGTTGGATTTATGACTATTAAAGTTAGTATACA1500
GCATACACAATCTATTGAAGGATATTTATAATGCAATTCCCTAAAAATAGTTTTGTATAA1560
CCAGTTCTTTTATCCGAACTGATACACGTATTTTAGCATAATTTTTAATGTATCTTCAAA1620
AACAGCTTCTGTGTCCTTTTCTATTAAACATATAAATTCTTTTTTATGTTATATATTTAT1680
AAAAGTTCTGTTTAAAAAGCCAAAAATAAATAATTATCTCTTTTTATTTATATTATATTG1740
AAACTAAAGTTTATTAATTTCAATATAATATAAATTTAATTTTATACAAAAAGGAGAACG1800
TATATGAAAAAACGAAAAGTGTTAATACCATTAATGGCATTGTCTACGATATTAGTTTCA1860
AGCACAGGTAATTTAGAGGTGATTCAGGCAGAAGTTAAACAGGAGAACCGGTTA1914
GluValLysGlnGluAsnArgLeu 15
TTAAATGAATCAGAATCAAGTTCCCAGGGGTTACTAGGATACTATTTT1962
LeuAsnGluSerGluSerSerSerGlnGlyLeuLeuGlyTyrTyrPhe 101520
AGTGATTTGAATTTTCAAGCACCCATGGTGGTTACCTCTTCTACTACA2010
SerAspLeuAsnPheGlnAlaProMetValValThrSerSerThrThr 25303540
GGGGATTTATCTATTCCTAGTTCTGAGTTAGAAAATATTCCATCGGAA2058
GlyAspLeuSerIleProSerSerGluLeuGluAsnIleProSerGlu 455055
AACCAATATTTTCAATCTGCTATTTGGTCAGGATTTATCAAAGTTAAG2106
AsnGlnTyrPheGlnSerAlaIleTrpSerGlyPheIleLysValLys 606570
AAGAGTGATGAATATACATTTGCTACTTCCGCTGATAATCATGTAACA2154
LysSerAspGluTyrThrPheAlaThrSerAlaAspAsnHisValThr 758085
ATGTGGGTAGATGACCAAGAAGTGATTAATAAAGCTTCTAATTCTAAC2202
MetTrpValAspAspGlnGluValIleAsnLysAlaSerAsnSerAsn 9095100
AAAATCAGATTAGAAAAAGGAAGATTATATCAAATAAAAATTCAATAT2250
LysIleArgLeuGluLysGlyArgLeuTyrGlnIleLysIleGlnTyr 105110115120
CAACGAGAAAATCCTACTGAAAAAGGATTGGATTTCAAGTTGTACTGG2298
GlnArgGluAsnProThrGluLysGlyLeuAspPheLysLeuTyrTrp 125130135
ACCGATTCTCAAAATAAAAAAGAAGTGATTTCTAGTGATAACTTACAA2346
ThrAspSerGlnAsnLysLysGluValIleSerSerAspAsnLeuGln 140145150
TTGCCAGAATTAAAACAAAAATCTTCGAACTCAAGAAAAAAGCGAAGT2394
LeuProGluLeuLysGlnLysSerSerAsnSerArgLysLysArgSer 155160165
ACAAGTGCTGGACCTACGGTTCCAGACCGTGACAATGATGGAATCCCT2442
ThrSerAlaGlyProThrValProAspArgAspAsnAspGlyIlePro 170175180
GATTCATTAGAGGTAGAAGGATATACGGTTGATGTCAAAAATAAAAGA2490
AspSerLeuGluValGluGlyTyrThrValAspValLysAsnLysArg 185190195200
ACTTTTCTTTCACCATGGATTTCTAATATTCATGAAAAGAAAGGATTA2538
ThrPheLeuSerProTrpIleSerAsnIleHisGluLysLysGlyLeu 205210215
ACCAAATATAAATCATCTCCTGAAAAATGGAGCACGGCTTCTGATCCG2586
ThrLysTyrLysSerSerProGluLysTrpSerThrAlaSerAspPro 220225230
TACAGTGATTTCGAAAAGGTTACAGGACGGATTGATAAGAATGTATCA2634
TyrSerAspPheGluLysValThrGlyArgIleAspLysAsnValSer 235240245
CCAGAGGCAAGACACCCCCTTGTGGCAGCTTATCCGATTGTACATGTA2682
ProGluAlaArgHisProLeuValAlaAlaTyrProIleValHisVal 250255260
GATATGGAGAATATTATTCTCTCAAAAAATGAGGATCAATCCACACAG2730
AspMetGluAsnIleIleLeuSerLysAsnGluAspGlnSerThrGln 265270275280
AATACTGATAGTGAAACGAGAACAATAAGTAAAAATACTTCTACAAGT2778
AsnThrAspSerGluThrArgThrIleSerLysAsnThrSerThrSer 285290295
AGGACACATACTAGTGAAGTACATGGAAATGCAGAAGTGCATGCGTCG2826
ArgThrHisThrSerGluValHisGlyAsnAlaGluValHisAlaSer 300305310
TTCTTTGATATTGGTGGGAGTGTATCTGCAGGATTTAGTAATTCGAAT2874
PhePheAspIleGlyGlySerValSerAlaGlyPheSerAsnSerAsn 315320325
TCAAGTACGGTCGCAATTGATCATTCACTATCTCTAGCAGGGGAAAGA2922
SerSerThrValAlaIleAspHisSerLeuSerLeuAlaGlyGluArg 330335340
ACTTGGGCTGAAACAATGGGTTTAAATACCGCTGATACAGCAAGATTA2970
ThrTrpAlaGluThrMetGlyLeuAsnThrAlaAspThrAlaArgLeu 345350355360
AATGCCAATATTAGATATGTAAATACTGGGACGGCTCCAATCTACAAC3018
AsnAlaAsnIleArgTyrValAsnThrGlyThrAlaProIleTyrAsn 365370375
GTGTTACCAACGACTTCGTTAGTGTTAGGAAAAAATCAAACACTCGCG3066
ValLeuProThrThrSerLeuValLeuGlyLysAsnGlnThrLeuAla 380385390
ACAATTAAAGCTAAGGAAAACCAATTAAGTCAAATACTTGCACCTAAT3114
ThrIleLysAlaLysGluAsnGlnLeuSerGlnIleLeuAlaProAsn 395400405
AATTATTATCCTTCTAAAAACTTGGCGCCAATCGCATTAAATGCACAA3162
AsnTyrTyrProSerLysAsnLeuAlaProIleAlaLeuAsnAlaGln 410415420
GACGATTTCAGTTCTACTCCAATTACAATGAATTACAATCAATTTCTT3210
AspAspPheSerSerThrProIleThrMetAsnTyrAsnGlnPheLeu 425430435440
GAGTTAGAAAAAACGAAACAATTAAGATTAGATACGGATCAAGTATAT3258
GluLeuGluLysThrLysGlnLeuArgLeuAspThrAspGlnValTyr 445450455
GGGAATATAGCAACATACAATTTTGAAAATGGAAGAGTGAGGGTGGAT3306
GlyAsnIleAlaThrTyrAsnPheGluAsnGlyArgValArgValAsp 460465470
ACAGGCTCGAACTGGAGTGAAGTGTTACCGCAAATTCAAGAAACAACT3354
ThrGlySerAsnTrpSerGluValLeuProGlnIleGlnGluThrThr 475480485
GCACGTATCATTTTTAATGGAAAAGATTTAAATCTGGTAGAAAGGCGG3402
AlaArgIleIlePheAsnGlyLysAspLeuAsnLeuValGluArgArg 490495500
ATAGCGGCGGTTAATCCTAGTGATCCATTAGAAACGACTAAACCGGAT3450
IleAlaAlaValAsnProSerAspProLeuGluThrThrLysProAsp 505510515520
ATGACATTAAAAGAAGCCCTTAAAATAGCATTTGGATTTAACGAACCG3498
MetThrLeuLysGluAlaLeuLysIleAlaPheGlyPheAsnGluPro 525530535
AATGGAAACTTACAATATCAAGGGAAAGACATAACCGAATTTGATTTT3546
AsnGlyAsnLeuGlnTyrGlnGlyLysAspIleThrGluPheAspPhe 540545550
AATTTCGATCAACAAACATCTCAAAATATCAAGAATCAGTTAGCGGAA3594
AsnPheAspGlnGlnThrSerGlnAsnIleLysAsnGlnLeuAlaGlu 555560565
TTAAACGCAACTAACATATATACTGTATTAGATAAAATCAAATTAAAT3642
LeuAsnAlaThrAsnIleTyrThrValLeuAspLysIleLysLeuAsn 570575580
GCAAAAATGAATATTTTAATAAGAGATAAACGTTTTCATTATGATAGA3690
AlaLysMetAsnIleLeuIleArgAspLysArgPheHisTyrAspArg 585590595600
AATAACATAGCAGTTGGGGCGGATGAGTCAGTAGTTAAGGAGGCTCAT3738
AsnAsnIleAlaValGlyAlaAspGluSerValValLysGluAlaHis 605610615
AGAGAAGTAATTAATTCGTCAACAGAGGGATTATTGTTAAATATTGAT3786
ArgGluValIleAsnSerSerThrGluGlyLeuLeuLeuAsnIleAsp 620625630
AAGGATATAAGAAAAATATTATCAGGTTATATTGTAGAAATTGAAGAT3834
LysAspIleArgLysIleLeuSerGlyTyrIleValGluIleGluAsp 635640645
ACTGAAGGGCTTAAAGAAGTTATAAATGACAGATATGATATGTTGAAT3882
ThrGluGlyLeuLysGluValIleAsnAspArgTyrAspMetLeuAsn 650655660
ATTTCTAGTTTACGGCAAGATGGAAAAACATTTATAGATTTTAAAAAA3930
IleSerSerLeuArgGlnAspGlyLysThrPheIleAspPheLysLys 665670675680
TATAATGATAAATTACCGTTATATATAAGTAATCCCAATTATAAGGTA3978
TyrAsnAspLysLeuProLeuTyrIleSerAsnProAsnTyrLysVal 685690695
AATGTATATGCTGTTACTAAAGAAAACACTATTATTAATCCTAGTGAG4026
AsnValTyrAlaValThrLysGluAsnThrIleIleAsnProSerGlu 700705710
AATGGGGATACTAGTACCAACGGGATCAAGAAAATTTTAATCTTTTCT4074
AsnGlyAspThrSerThrAsnGlyIleLysLysIleLeuIlePheSer 715720725
AAAAAAGGCTATGAGATAGGATAAGGTAATTCTAGGTGATTTTTAAATTAT4125
LysLysGlyTyrGluIleGly 730735
CTAAAAAACAGTAAAATTAAAACATACTCTTTTTGTAAGAAATACAAGGAGAGTATGTTT4185
TAAACAGTAATCTAAATCATCATAATCCTTTGAGATTGTTTGTAGGATCC4235 (2)
INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 735 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GluValLysGlnGluAsnArgLeuLeuAsnGluSerGluSerSerSer 151015
GlnGlyLeuLeuGlyTyrTyrPheSerAspLeuAsnPheGlnAlaPro 202530
MetValValThrSerSerThrThrGlyAspLeuSerIleProSerSer 354045
GluLeuGluAsnIleProSerGluAsnGlnTyrPheGlnSerAlaIle 505560
TrpSerGlyPheIleLysValLysLysSerAspGluTyrThrPheAla 65707580
ThrSerAlaAspAsnHisValThrMetTrpValAspAspGlnGluVal 859095
IleAsnLysAlaSerAsnSerAsnLysIleArgLeuGluLysGlyArg 100105110
LeuTyrGlnIleLysIleGlnTyrGlnArgGluAsnProThrGluLys
115120125 GlyLeuAspPheLysLeuTyrTrpThrAspSerGlnAsnLysLysGlu
130135140 ValIleSerSerAspAsnLeuGlnLeuProGluLeuLysGlnLysSer
145150155160 SerAsnSerArgLysLysArgSerThrSerAlaGlyProThrValPro
165170175 AspArgAspAsnAspGlyIleProAspSerLeuGluValGluGlyTyr
180185190 ThrValAspValLysAsnLysArgThrPheLeuSerProTrpIleSer
195200205 AsnIleHisGluLysLysGlyLeuThrLysTyrLysSerSerProGlu
210215220 LysTrpSerThrAlaSerAspProTyrSerAspPheGluLysValThr
225230235240 GlyArgIleAspLysAsnValSerProGluAlaArgHisProLeuVal
245250255 AlaAlaTyrProIleValHisValAspMetGluAsnIleIleLeuSer
260265270 LysAsnGluAspGlnSerThrGlnAsnThrAspSerGluThrArgThr
275280285 IleSerLysAsnThrSerThrSerArgThrHisThrSerGluValHis
290295300 GlyAsnAlaGluValHisAlaSerPhePheAspIleGlyGlySerVal
305310315320 SerAlaGlyPheSerAsnSerAsnSerSerThrValAlaIleAspHis
325330335 SerLeuSerLeuAlaGlyGluArgThrTrpAlaGluThrMetGlyLeu
340345350 AsnThrAlaAspThrAlaArgLeuAsnAlaAsnIleArgTyrValAsn
355360365 ThrGlyThrAlaProIleTyrAsnValLeuProThrThrSerLeuVal
370375380 LeuGlyLysAsnGlnThrLeuAlaThrIleLysAlaLysGluAsnGln
385390395400 LeuSerGlnIleLeuAlaProAsnAsnTyrTyrProSerLysAsnLeu
405410415 AlaProIleAlaLeuAsnAlaGlnAspAspPheSerSerThrProIle
420425430 ThrMetAsnTyrAsnGlnPheLeuGluLeuGluLysThrLysGlnLeu
435440445 ArgLeuAspThrAspGlnValTyrGlyAsnIleAlaThrTyrAsnPhe
450455460 GluAsnGlyArgValArgValAspThrGlySerAsnTrpSerGluVal
465470475480 LeuProGlnIleGlnGluThrThrAlaArgIleIlePheAsnGlyLys
485490495 AspLeuAsnLeuValGluArgArgIleAlaAlaValAsnProSerAsp
500505510 ProLeuGluThrThrLysProAspMetThrLeuLysGluAlaLeuLys
515520525 IleAlaPheGlyPheAsnGluProAsnGlyAsnLeuGlnTyrGlnGly
530535540 LysAspIleThrGluPheAspPheAsnPheAspGlnGlnThrSerGln
545550555560 AsnIleLysAsnGlnLeuAlaGluLeuAsnAlaThrAsnIleTyrThr
565570575 ValLeuAspLysIleLysLeuAsnAlaLysMetAsnIleLeuIleArg
580585590 AspLysArgPheHisTyrAspArgAsnAsnIleAlaValGlyAlaAsp
595600605 GluSerValValLysGluAlaHisArgGluValIleAsnSerSerThr
610615620 GluGlyLeuLeuLeuAsnIleAspLysAspIleArgLysIleLeuSer
625630635640 GlyTyrIleValGluIleGluAspThrGluGlyLeuLysGluValIle
645650655 AsnAspArgTyrAspMetLeuAsnIleSerSerLeuArgGlnAspGly
660665670 LysThrPheIleAspPheLysLysTyrAsnAspLysLeuProLeuTyr
675680685 IleSerAsnProAsnTyrLysValAsnValTyrAlaValThrLysGlu
690695700 AsnThrIleIleAsnProSerGluAsnGlyAspThrSerThrAsnGly
705710715720 IleLysLysIleLeuIlePheSerLysLysGlyTyrGluIleGly
725730735 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 1368 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1..1368 (D) OTHER INFORMATION:
/product="LF(1-254)--TR--PE(401-602)" (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:5: GCGGGCGGTCATGGTGATGTAGGTATGCACGTAAAAGAGAAAGAGAAA48
AlaGlyGlyHisGlyAspValGlyMetHisValLysGluLysGluLys 151015
AATAAAGATGAGAATAAGAGAAAAGATGAAGAACGAAATAAAACACAG96
AsnLysAspGluAsnLysArgLysAspGluGluArgAsnLysThrGln 202530
GAAGAGCATTTAAAGGAAATCATGAAACACATTGTAAAAATAGAAGTA144
GluGluHisLeuLysGluIleMetLysHisIleValLysIleGluVal 354045
AAAGGGGAGGAAGCTGTTAAAAAAGAGGCAGCAGAAAAGCTACTTGAG192
LysGlyGluGluAlaValLysLysGluAlaAlaGluLysLeuLeuGlu 505560
AAAGTACCATCTGATGTTTTAGAGATGTATAAAGCAATTGGAGGAAAG240
LysValProSerAspValLeuGluMetTyrLysAlaIleGlyGlyLys 65707580
ATATATATTGTGGATGGTGATATTACAAAACATATATCTTTAGAAGCA288
IleTyrIleValAspGlyAspIleThrLysHisIleSerLeuGluAla 859095
TTATCTGAAGATAAGAAAAAAATAAAAGACATTTATGGGAAAGATGCT336
LeuSerGluAspLysLysLysIleLysAspIleTyrGlyLysAspAla 100105110
TTATTACATGAACATTATGTATATGCAAAAGAAGGATATGAACCCGTA384
LeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyrGluProVal 115120125
CTTGTAATCCAATCTTCGGAAGATTATGTAGAAAATACTGAAAAGGCA432
LeuValIleGlnSerSerGluAspTyrValGluAsnThrGluLysAla 130135140
CTGAACGTTTATTATGAAATAGGTAAGATATTATCAAGGGATATTTTA480
LeuAsnValTyrTyrGluIleGlyLysIleLeuSerArgAspIleLeu 145150155160
AGTAAAATTAATCAACCATATCAGAAATTTTTAGATGTATTAAATACC528
SerLysIleAsnGlnProTyrGlnLysPheLeuAspValLeuAsnThr 165170175
ATTAAAAATGCATCTGATTCAGATGGACAAGATCTTTTATTTACTAAT576
IleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeuPheThrAsn 180185190
CAGCTTAAGGAACATCCCACAGACTTTTCTGTAGAATTCTTGGAACAA624
GlnLeuLysGluHisProThrAspPheSerValGluPheLeuGluGln 195200205
AATAGCAATGAGGTACAAGAAGTATTTGCGAAAGCTTTTGCATATTAT672
AsnSerAsnGluValGlnGluValPheAlaLysAlaPheAlaTyrTyr 210215220
ATCGAGCCACAGCATCGTGATGTTTTACAGCTTTATGCACCGGAAGCT720
IleGluProGlnHisArgAspValLeuGlnLeuTyrAlaProGluAla 225230235240
TTTAATTACATGGATAAATTTAACGAACAAGAAATAAATCTACTCGGC768
PheAsnTyrMetAspLysPheAsnGluGlnGluIleAsnLeuLeuGly 245250255
GACGGCGGCGACGTCAGCTTCAGCACCCGCGGCACGCAGAACTGGACG816
AspGlyGlyAspValSerPheSerThrArgGlyThrGlnAsnTrpThr 260265270
GTGGAGCGGCTGCTCCAGGCGCACCGCCAACTGGAGGAGCGCGGCTAT864
ValGluArgLeuLeuGlnAlaHisArgGlnLeuGluGluArgGlyTyr 275280285
GTGTTCGTCGGCTACCACGGCACCTTCCTCGAAGCGGCGCAAAGCATC912
ValPheValGlyTyrHisGlyThrPheLeuGluAlaAlaGlnSerIle 290295300
GTCTTCGGCGGGGTGCGCGCGCGCAGCCAGGACCTCGACGCGATCTGG960
ValPheGlyGlyValArgAlaArgSerGlnAspLeuAspAlaIleTrp 305310315320
CGCGGTTTCTATATCGCCGGCGATCCGGCGCTGGCCTACGGCTACGCC1008
ArgGlyPheTyrIleAlaGlyAspProAlaLeuAlaTyrGlyTyrAla 325330335
CAGGACCAGGAACCCGACGCACGCGGCCGGATCCGCAACGGTGCCCTG1056
GlnAspGlnGluProAspAlaArgGlyArgIleArgAsnGlyAlaLeu 340345350
CTGCGGGTCTATGTGCCGCGCTCGAGCCTGCCGGGCTTCTACCGCACC1104
LeuArgValTyrValProArgSerSerLeuProGlyPheTyrArgThr 355360365
AGCCTGACCCTGGCCGCGCCGGAGGCGGCGGGCGAGGTCGAACGGCTG1152
SerLeuThrLeuAlaAlaProGluAlaAlaGlyGluValGluArgLeu 370375380
ATCGGCCATCCGCTGCCGCTGCGCCTGGACGCCATCACCGGCCCCGAG1200
IleGlyHisProLeuProLeuArgLeuAspAlaIleThrGlyProGlu 385390395400
GAGGAAGGCGGGCGCCTGGAGACCATTCTCGGCTGGCCGCTGGCCGAG1248
GluGluGlyGlyArgLeuGluThrIleLeuGlyTrpProLeuAlaGlu 405410415
CGCACCGTGGTGATTCCCTCGGCGATCCCCACCGACCCGCGCAACGTC1296
ArgThrValValIleProSerAlaIleProThrAspProArgAsnVal 420425430
GGCGGCGACCTCGACCCGTCCAGCATCCCCGACAAGGAACAGGCGATC1344
GlyGlyAspLeuAspProSerSerIleProAspLysGluGlnAlaIle 435440445
AGCGCCCTGCCGGACTACGCCAGC1368 SerAlaLeuProAspTyrAlaSer 450455 (2)
INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 456 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
AlaGlyGlyHisGlyAspValGlyMetHisValLysGluLysGluLys 151015
AsnLysAspGluAsnLysArgLysAspGluGluArgAsnLysThrGln 202530
GluGluHisLeuLysGluIleMetLysHisIleValLysIleGluVal 354045
LysGlyGluGluAlaValLysLysGluAlaAlaGluLysLeuLeuGlu 505560
LysValProSerAspValLeuGluMetTyrLysAlaIleGlyGlyLys 65707580
IleTyrIleValAspGlyAspIleThrLysHisIleSerLeuGluAla 859095
LeuSerGluAspLysLysLysIleLysAspIleTyrGlyLysAspAla 100105110
LeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyrGluProVal 115120125
LeuValIleGlnSerSerGluAspTyrValGluAsnThrGluLysAla 130135140
LeuAsnValTyrTyrGluIleGlyLysIleLeuSerArgAspIleLeu 145150155160
SerLysIleAsnGlnProTyrGlnLysPheLeuAspValLeuAsnThr 165170175
IleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeuPheThrAsn 180185190
GlnLeuLysGluHisProThrAspPheSerValGluPheLeuGluGln 195200205
AsnSerAsnGluValGlnGluValPheAlaLysAlaPheAlaTyrTyr 210215220
IleGluProGlnHisArgAspValLeuGlnLeuTyrAlaProGluAla 225230235240
PheAsnTyrMetAspLysPheAsnGluGlnGluIleAsnLeuLeuGly 245250255
AspGlyGlyAspValSerPheSerThrArgGlyThrGlnAsnTrpThr 260265270
ValGluArgLeuLeuGlnAlaHisArgGlnLeuGluGluArgGlyTyr 275280285
ValPheValGlyTyrHisGlyThrPheLeuGluAlaAlaGlnSerIle 290295300
ValPheGlyGlyValArgAlaArgSerGlnAspLeuAspAlaIleTrp 305310315320
ArgGlyPheTyrIleAlaGlyAspProAlaLeuAlaTyrGlyTyrAla 325330335
GlnAspGlnGluProAspAlaArgGlyArgIleArgAsnGlyAlaLeu 340345350
LeuArgValTyrValProArgSerSerLeuProGlyPheTyrArgThr 355360365
SerLeuThrLeuAlaAlaProGluAlaAlaGlyGluValGluArgLeu 370375380
IleGlyHisProLeuProLeuArgLeuAspAlaIleThrGlyProGlu 385390395400
GluGluGlyGlyArgLeuGluThrIleLeuGlyTrpProLeuAlaGlu 405410415
ArgThrValValIleProSerAlaIleProThrAspProArgAsnVal 420425430
GlyGlyAspLeuAspProSerSerIleProAspLysGluGlnAlaIle 435440445
SerAlaLeuProAspTyrAlaSer 450455 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1425 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1..1416 (D) OTHER INFORMATION:
/product="LF(1-254)--TR--PE(398-613)" (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:7: ATGGTACCAGCGGGCGGTCATGGTGATGTAGGTATGCACGTAAAAGAG48
MetValProAlaGlyGlyHisGlyAspValGlyMetHisValLysGlu 151015
AAAGAGAAAAATAAAGATGAGAATAAGAGAAAAGATGAAGAACGAAAT96
LysGluLysAsnLysAspGluAsnLysArgLysAspGluGluArgAsn 202530
AAAACACAGGAAGAGCATTTAAAGGAAATCATGAAACACATTGTAAAA144
LysThrGlnGluGluHisLeuLysGluIleMetLysHisIleValLys 354045
ATAGAAGTAAAAGGGGAGGAAGCTGTTAAAAAAGAGGCAGCAGAAAAG192
IleGluValLysGlyGluGluAlaValLysLysGluAlaAlaGluLys 505560
CTACTTGAGAAAGTACCATCTGATGTTTTAGAGATGTATAAAGCAATT240
LeuLeuGluLysValProSerAspValLeuGluMetTyrLysAlaIle 65707580
GGAGGAAAGATATATATTGTGGATGGTGATATTACAAAACATATATCT288
GlyGlyLysIleTyrIleValAspGlyAspIleThrLysHisIleSer 859095
TTAGAAGCATTATCTGAAGATAAGAAAAAAATAAAAGACATTTATGGG336
LeuGluAlaLeuSerGluAspLysLysLysIleLysAspIleTyrGly 100105110
AAAGATGCTTTATTACATGAACATTATGTATATGCAAAAGAAGGATAT384
LysAspAlaLeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyr 115120125
GAACCCGTACTTGTAATCCAATCTTCGGAAGATTATGTAGAAAATACT432
GluProValLeuValIleGlnSerSerGluAspTyrValGluAsnThr 130135140
GAAAAGGCACTGAACGTTTATTATGAAATAGGTAAGATATTATCAAGG480
GluLysAlaLeuAsnValTyrTyrGluIleGlyLysIleLeuSerArg 145150155160
GATATTTTAAGTAAAATTAATCAACCATATCAGAAATTTTTAGATGTA528
AspIleLeuSerLysIleAsnGlnProTyrGlnLysPheLeuAspVal 165170175
TTAAATACCATTAAAAATGCATCTGATTCAGATGGACAAGATCTTTTA576
LeuAsnThrIleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeu 180185190
TTTACTAATCAGCTTAAGGAACATCCCACAGACTTTTCTGTAGAATTC624
PheThrAsnGlnLeuLysGluHisProThrAspPheSerValGluPhe 195200205
TTGGAACAAAATAGCAATGAGGTACAAGAAGTATTTGCGAAAGCTTTT672
LeuGluGlnAsnSerAsnGluValGlnGluValPheAlaLysAlaPhe 210215220
GCATATTATATCGAGCCACAGCATCGTGATGTTTTACAGCTTTATGCA720
AlaTyrTyrIleGluProGlnHisArgAspValLeuGlnLeuTyrAla 225230235240
CCGGAAGCTTTTAATTACATGGATAAATTTAACGAACAAGAAATAAAT768
ProGluAlaPheAsnTyrMetAspLysPheAsnGluGlnGluIleAsn 245250255
CTAACGCGTGCGGAGTTCCTCGGCGACGGCGGCGACGTCAGCTTCAGC816
LeuThrArgAlaGluPheLeuGlyAspGlyGlyAspValSerPheSer 260265270
ACCCGCGGCACGCAGAACTGGACGGTGGAGCGGCTGCTCCAGGCGCAC864
ThrArgGlyThrGlnAsnTrpThrValGluArgLeuLeuGlnAlaHis 275280285
CGCCAACTGGAGGAGCGCGGCTATGTGTTCGTCGGCTACCACGGCACC912
ArgGlnLeuGluGluArgGlyTyrValPheValGlyTyrHisGlyThr 290295300
TTCCTCGAAGCGGCGCAAAGCATCGTCTTCGGCGGGGTGCGCGCGCGC960
PheLeuGluAlaAlaGlnSerIleValPheGlyGlyValArgAlaArg 305310315320
AGCCAGGACCTCGACGCGATCTGGCGCGGTTTCTATATCGCCGGCGAT1008
SerGlnAspLeuAspAlaIleTrpArgGlyPheTyrIleAlaGlyAsp 325330335
CCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCGACGCACGC1056
ProAlaLeuAlaTyrGlyTyrAlaGlnAspGlnGluProAspAlaArg 340345350
GGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGCTCG1104
GlyArgIleArgAsnGlyAlaLeuLeuArgValTyrValProArgSer 355360365
AGCCTGCCGGGCTTCTACCGCACCAGCCTGACCCTGGCCGCGCCGGAG1152
SerLeuProGlyPheTyrArgThrSerLeuThrLeuAlaAlaProGlu 370375380
GCGGCGGGCGAGGTCGAACGGCTGATCGGCCATCCGCTGCCGCTGCGC1200
AlaAlaGlyGluValGluArgLeuIleGlyHisProLeuProLeuArg 385390395400
CTGGACGCCATCACCGGCCCCGAGGAGGAAGGCGGGCGCCTGGAGACC1248
LeuAspAlaIleThrGlyProGluGluGluGlyGlyArgLeuGluThr 405410415
ATTCTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTCCCTCGGCG1296
IleLeuGlyTrpProLeuAlaGluArgThrValValIleProSerAla 420425430
ATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCGACCCGTCCAGC1344
IleProThrAspProArgAsnValGlyGlyAspLeuAspProSerSer 435440445
ATCCCCGACAAGGAACAGGCGATCAGCGCCCTGCCGGACTACGCCAGC1392
IleProAspLysGluGlnAlaIleSerAlaLeuProAspTyrAlaSer 450455460
CAGCCCGGCAAACCGCCGCGCGAGGACCTGAAG1425 GlnProGlyLysProProArgGlu
465470 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 472 amino acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:8:
MetValProAlaGlyGlyHisGlyAspValGlyMetHisValLysGlu 151015
LysGluLysAsnLysAspGluAsnLysArgLysAspGluGluArgAsn 202530
LysThrGlnGluGluHisLeuLysGluIleMetLysHisIleValLys 354045
IleGluValLysGlyGluGluAlaValLysLysGluAlaAlaGluLys 505560
LeuLeuGluLysValProSerAspValLeuGluMetTyrLysAlaIle 65707580
GlyGlyLysIleTyrIleValAspGlyAspIleThrLysHisIleSer 859095
LeuGluAlaLeuSerGluAspLysLysLysIleLysAspIleTyrGly 100105110
LysAspAlaLeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyr 115120125
GluProValLeuValIleGlnSerSerGluAspTyrValGluAsnThr 130135140
GluLysAlaLeuAsnValTyrTyrGluIleGlyLysIleLeuSerArg 145150155160
AspIleLeuSerLysIleAsnGlnProTyrGlnLysPheLeuAspVal 165170175
LeuAsnThrIleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeu 180185190
PheThrAsnGlnLeuLysGluHisProThrAspPheSerValGluPhe 195200205
LeuGluGlnAsnSerAsnGluValGlnGluValPheAlaLysAlaPhe 210215220
AlaTyrTyrIleGluProGlnHisArgAspValLeuGlnLeuTyrAla 225230235240
ProGluAlaPheAsnTyrMetAspLysPheAsnGluGlnGluIleAsn 245250255
LeuThrArgAlaGluPheLeuGlyAspGlyGlyAspValSerPheSer 260265270
ThrArgGlyThrGlnAsnTrpThrValGluArgLeuLeuGlnAlaHis 275280285
ArgGlnLeuGluGluArgGlyTyrValPheValGlyTyrHisGlyThr 290295300
PheLeuGluAlaAlaGlnSerIleValPheGlyGlyValArgAlaArg 305310315320
SerGlnAspLeuAspAlaIleTrpArgGlyPheTyrIleAlaGlyAsp 325330335
ProAlaLeuAlaTyrGlyTyrAlaGlnAspGlnGluProAspAlaArg 340345350
GlyArgIleArgAsnGlyAlaLeuLeuArgValTyrValProArgSer 355360365
SerLeuProGlyPheTyrArgThrSerLeuThrLeuAlaAlaProGlu 370375380
AlaAlaGlyGluValGluArgLeuIleGlyHisProLeuProLeuArg 385390395400
LeuAspAlaIleThrGlyProGluGluGluGlyGlyArgLeuGluThr 405410415
IleLeuGlyTrpProLeuAlaGluArgThrValValIleProSerAla 420425430
IleProThrAspProArgAsnValGlyGlyAspLeuAspProSerSer 435440445
IleProAspLysGluGlnAlaIleSerAlaLeuProAspTyrAlaSer 450455460
GlnProGlyLysProProArgGlu 465470 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1524 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL
SOURCE: (A) ORGANISM: Bacillus anthracis (ix) FEATURE: (A)
NAME/KEY: CDS (B) LOCATION: 1..1524 (D) OTHER INFORMATION:
/product="LF(1-254)--TR--PE(362-613)" (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:9: GCGGGCGGTCATGGTGATGTAGGTATGCACGTAAAAGAGAAAGAGAAA48
AlaGlyGlyHisGlyAspValGlyMetHisValLysGluLysGluLys 151015
AATAAAGATGAGAATAAGAGAAAAGATGAAGAACGAAATAAAACACAG96
AsnLysAspGluAsnLysArgLysAspGluGluArgAsnLysThrGln 202530
GAAGAGCATTTAAAGGAAATCATGAAACACATTGTAAAAATAGAAGTA144
GluGluHisLeuLysGluIleMetLysHisIleValLysIleGluVal 354045
AAAGGGGAGGAAGCTGTTAAAAAAGAGGCAGCAGAAAAGCTACTTGAG192
LysGlyGluGluAlaValLysLysGluAlaAlaGluLysLeuLeuGlu 505560
AAAGTACCATCTGATGTTTTAGAGATGTATAAAGCAATTGGAGGAAAG240
LysValProSerAspValLeuGluMetTyrLysAlaIleGlyGlyLys 65707580
ATATATATTGTGGATGGTGATATTACAAAACATATATCTTTAGAAGCA288
IleTyrIleValAspGlyAspIleThrLysHisIleSerLeuGluAla 859095
TTATCTGAAGATAAGAAAAAAATAAAAGACATTTATGGGAAAGATGCT336
LeuSerGluAspLysLysLysIleLysAspIleTyrGlyLysAspAla 100105110
TTATTACATGAACATTATGTATATGCAAAAGAAGGATATGAACCCGTA384
LeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyrGluProVal 115120125
CTTGTAATCCAATCTTCGGAAGATTATGTAGAAAATACTGAAAAGGCA432
LeuValIleGlnSerSerGluAspTyrValGluAsnThrGluLysAla 130135140
CTGAACGTTTATTATGAAATAGGTAAGATATTATCAAGGGATATTTTA480
LeuAsnValTyrTyrGluIleGlyLysIleLeuSerArgAspIleLeu 145150155160
AGTAAAATTAATCAACCATATCAGAAATTTTTAGATGTATTAAATACC528
SerLysIleAsnGlnProTyrGlnLysPheLeuAspValLeuAsnThr 165170175
ATTAAAAATGCATCTGATTCAGATGGACAAGATCTTTTATTTACTAAT576
IleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeuPheThrAsn 180185190
CAGCTTAAGGAACATCCCACAGACTTTTCTGTAGAATTCTTGGAACAA624
GlnLeuLysGluHisProThrAspPheSerValGluPheLeuGluGln 195200205
AATAGCAATGAGGTACAAGAAGTATTTGCGAAAGCTTTTGCATATTAT672
AsnSerAsnGluValGlnGluValPheAlaLysAlaPheAlaTyrTyr 210215220
ATCGAGCCACAGCATCGTGATGTTTTACAGCTTTATGCACCGGAAGCT720
IleGluProGlnHisArgAspValLeuGlnLeuTyrAlaProGluAla 225230235240
TTTAATTACATGGATAAATTTAACGAACAAGAAATAAATCTAACGCGT768
PheAsnTyrMetAspLysPheAsnGluGlnGluIleAsnLeuThrArg 245250255
GCGGCCAACGCCGACGTGGTGAGCCTGACCTGCCCGGTCGCCGCCGGT816
AlaAlaAsnAlaAspValValSerLeuThrCysProValAlaAlaGly 260265270
GAATGCGCGGGCCCGGCGGACAGCGGCGACGCCCTGCTGGAGCGCAAC864
GluCysAlaGlyProAlaAspSerGlyAspAlaLeuLeuGluArgAsn 275280285
TATCCCACTGGCGCGGAGTTCCTCGGCGACGGCGGCGACGTCAGCTTC912
TyrProThrGlyAlaGluPheLeuGlyAspGlyGlyAspValSerPhe 290295300
AGCACCCGCGGCACGCAGAACTGGACGGTGGAGCGGCTGCTCCAGGCG960
SerThrArgGlyThrGlnAsnTrpThrValGluArgLeuLeuGlnAla 305310315320
CACCGCCAACTGGAGGAGCGCGGCTATGTGTTCGTCGGCTACCACGGC1008
HisArgGlnLeuGluGluArgGlyTyrValPheValGlyTyrHisGly 325330335
ACCTTCCTCGAAGCGGCGCAAAGCATCGTCTTCGGCGGGGTGCGCGCG1056
ThrPheLeuGluAlaAlaGlnSerIleValPheGlyGlyValArgAla 340345350
CGCAGCCAGGACCTCGACGCGATCTGGCGCGGTTTCTATATCGCCGGC1104
ArgSerGlnAspLeuAspAlaIleTrpArgGlyPheTyrIleAlaGly 355360365
GATCCGGCGCTGGCCTACGGCTACGCCCAGGACCAGGAACCCGACGCA1152
AspProAlaLeuAlaTyrGlyTyrAlaGlnAspGlnGluProAspAla 370375380
CGCGGCCGGATCCGCAACGGTGCCCTGCTGCGGGTCTATGTGCCGCGC1200
ArgGlyArgIleArgAsnGlyAlaLeuLeuArgValTyrValProArg 385390395400
TCGAGCCTGCCGGGCTTCTACCGCACCAGCCTGACCCTGGCCGCGCCG1248
SerSerLeuProGlyPheTyrArgThrSerLeuThrLeuAlaAlaPro 405410415
GAGGCGGCGGGCGAGGTCGAACGGCTGATCGGCCATCCGCTGCCGCTG1296
GluAlaAlaGlyGluValGluArgLeuIleGlyHisProLeuProLeu 420425430
CGCCTGGACGCCATCACCGGCCCCGAGGAGGAAGGCGGGCGCCTGGAG1344
ArgLeuAspAlaIleThrGlyProGluGluGluGlyGlyArgLeuGlu 435440445
ACCATTCTCGGCTGGCCGCTGGCCGAGCGCACCGTGGTGATTCCCTCG1392
ThrIleLeuGlyTrpProLeuAlaGluArgThrValValIleProSer 450455460
GCGATCCCCACCGACCCGCGCAACGTCGGCGGCGACCTCGACCCGTCC1440
AlaIleProThrAspProArgAsnValGlyGlyAspLeuAspProSer 465470475480
AGCATCCCCGACAAGGAACAGGCGATCAGCGCCCTGCCGGACTACGCC1488
SerIleProAspLysGluGlnAlaIleSerAlaLeuProAspTyrAla 485490495
AGCCAGCCCGGCAAACCGCCGCGCGAGGACCTGAAG1524
SerGlnProGlyLysProProArgGluAspLeuLys 500505 (2) INFORMATION FOR SEQ
ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 508 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
AlaGlyGlyHisGlyAspValGlyMetHisValLysGluLysGluLys 151015
AsnLysAspGluAsnLysArgLysAspGluGluArgAsnLysThrGln 202530
GluGluHisLeuLysGluIleMetLysHisIleValLysIleGluVal 354045
LysGlyGluGluAlaValLysLysGluAlaAlaGluLysLeuLeuGlu 505560
LysValProSerAspValLeuGluMetTyrLysAlaIleGlyGlyLys 65707580
IleTyrIleValAspGlyAspIleThrLysHisIleSerLeuGluAla 859095
LeuSerGluAspLysLysLysIleLysAspIleTyrGlyLysAspAla 100105110
LeuLeuHisGluHisTyrValTyrAlaLysGluGlyTyrGluProVal 115120125
LeuValIleGlnSerSerGluAspTyrValGluAsnThrGluLysAla 130135140
LeuAsnValTyrTyrGluIleGlyLysIleLeuSerArgAspIleLeu 145150155160
SerLysIleAsnGlnProTyrGlnLysPheLeuAspValLeuAsnThr 165170175
IleLysAsnAlaSerAspSerAspGlyGlnAspLeuLeuPheThrAsn 180185190
GlnLeuLysGluHisProThrAspPheSerValGluPheLeuGluGln 195200205
AsnSerAsnGluValGlnGluValPheAlaLysAlaPheAlaTyrTyr 210215220
IleGluProGlnHisArgAspValLeuGlnLeuTyrAlaProGluAla 225230235240
PheAsnTyrMetAspLysPheAsnGluGlnGluIleAsnLeuThrArg 245250255
AlaAlaAsnAlaAspValValSerLeuThrCysProValAlaAlaGly 260265270
GluCysAlaGlyProAlaAspSerGlyAspAlaLeuLeuGluArgAsn 275280285
TyrProThrGlyAlaGluPheLeuGlyAspGlyGlyAspValSerPhe 290295300
SerThrArgGlyThrGlnAsnTrpThrValGluArgLeuLeuGlnAla 305310315320
HisArgGlnLeuGluGluArgGlyTyrValPheValGlyTyrHisGly 325330335
ThrPheLeuGluAlaAlaGlnSerIleValPheGlyGlyValArgAla 340345350
ArgSerGlnAspLeuAspAlaIleTrpArgGlyPheTyrIleAlaGly 355360365
AspProAlaLeuAlaTyrGlyTyrAlaGlnAspGlnGluProAspAla 370375380
ArgGlyArgIleArgAsnGlyAlaLeuLeuArgValTyrValProArg 385390395400
SerSerLeuProGlyPheTyrArgThrSerLeuThrLeuAlaAlaPro 405410415
GluAlaAlaGlyGluValGluArgLeuIleGlyHisProLeuProLeu 420425430
ArgLeuAspAlaIleThrGlyProGluGluGluGlyGlyArgLeuGlu 435440445
ThrIleLeuGlyTrpProLeuAlaGluArgThrValValIleProSer 450455460
AlaIleProThrAspProArgAsnValGlyGlyAspLeuAspProSer 465470475480
SerIleProAspLysGluGlnAlaIleSerAlaLeuProAspTyrAla 485490495
SerGlnProGlyLysProProArgGluAspLeuLys 500505 (2) INFORMATION FOR SEQ
ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2709 base
pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2709 (D) OTHER
INFORMATION: /product="PA(1-725)-Human CD4 residues (1- 178)" (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:11:
GAAGTTAAACAGGAGAACCGGTTATTAAATGAATCAGAATCAAGTTCC48
GluValLysGlnGluAsnArgLeuLeuAsnGluSerGluSerSerSer 151015
CAGGGGTTACTAGGATACTATTTTAGTGATTTGAATTTTCAAGCACCC96
GlnGlyLeuLeuGlyTyrTyrPheSerAspLeuAsnPheGlnAlaPro 202530
ATGGTGGTTACCTCTTCTACTACAGGGGATTTATCTATTCCTAGTTCT144
MetValValThrSerSerThrThrGlyAspLeuSerIleProSerSer 354045
GAGTTAGAAAATATTCCATCGGAAAACCAATATTTTCAATCTGCTATT192
GluLeuGluAsnIleProSerGluAsnGlnTyrPheGlnSerAlaIle 505560
TGGTCAGGATTTATCAAAGTTAAGAAGAGTGATGAATATACATTTGCT240
TrpSerGlyPheIleLysValLysLysSerAspGluTyrThrPheAla 65707580
ACTTCCGCTGATAATCATGTAACAATGTGGGTAGATGACCAAGAAGTG288
ThrSerAlaAspAsnHisValThrMetTrpValAspAspGlnGluVal 859095
ATTAATAAAGCTTCTAATTCTAACAAAATCAGATTAGAAAAAGGAAGA336
IleAsnLysAlaSerAsnSerAsnLysIleArgLeuGluLysGlyArg 100105110
TTATATCAAATAAAAATTCAATATCAACGAGAAAATCCTACTGAAAAA384
LeuTyrGlnIleLysIleGlnTyrGlnArgGluAsnProThrGluLys 115120125
GGATTGGATTTCAAGTTGTACTGGACCGATTCTCAAAATAAAAAAGAA432
GlyLeuAspPheLysLeuTyrTrpThrAspSerGlnAsnLysLysGlu 130135140
GTGATTTCTAGTGATAACTTACAATTGCCAGAATTAAAACAAAAATCT480
ValIleSerSerAspAsnLeuGlnLeuProGluLeuLysGlnLysSer 145150155160
TCGAACTCAAGAAAAAAGCGAAGTACAAGTGCTGGACCTACGGTTCCA528
SerAsnSerArgLysLysArgSerThrSerAlaGlyProThrValPro 165170175
GACCGTGACAATGATGGAATCCCTGATTCATTAGAGGTAGAAGGATAT576
AspArgAspAsnAspGlyIleProAspSerLeuGluValGluGlyTyr 180185190
ACGGTTGATGTCAAAAATAAAAGAACTTTTCTTTCACCATGGATTTCT624
ThrValAspValLysAsnLysArgThrPheLeuSerProTrpIleSer 195200205
AATATTCATGAAAAGAAAGGATTAACCAAATATAAATCATCTCCTGAA672
AsnIleHisGluLysLysGlyLeuThrLysTyrLysSerSerProGlu 210215220
AAATGGAGCACGGCTTCTGATCCGTACAGTGATTTCGAAAAGGTTACA720
LysTrpSerThrAlaSerAspProTyrSerAspPheGluLysValThr 225230235240
GGACGGATTGATAAGAATGTATCACCAGAGGCAAGACACCCCCTTGTG768
GlyArgIleAspLysAsnValSerProGluAlaArgHisProLeuVal 245250255
GCAGCTTATCCGATTGTACATGTAGATATGGAGAATATTATTCTCTCA816
AlaAlaTyrProIleValHisValAspMetGluAsnIleIleLeuSer 260265270
AAAAATGAGGATCAATCCACACAGAATACTGATAGTGAAACGAGAACA864
LysAsnGluAspGlnSerThrGlnAsnThrAspSerGluThrArgThr 275280285
ATAAGTAAAAATACTTCTACAAGTAGGACACATACTAGTGAAGTACAT912
IleSerLysAsnThrSerThrSerArgThrHisThrSerGluValHis 290295300
GGAAATGCAGAAGTGCATGCGTCGTTCTTTGATATTGGTGGGAGTGTA960
GlyAsnAlaGluValHisAlaSerPhePheAspIleGlyGlySerVal 305310315320
TCTGCAGGATTTAGTAATTCGAATTCAAGTACGGTCGCAATTGATCAT1008
SerAlaGlyPheSerAsnSerAsnSerSerThrValAlaIleAspHis 325330335
TCACTATCTCTAGCAGGGGAAAGAACTTGGGCTGAAACAATGGGTTTA1056
SerLeuSerLeuAlaGlyGluArgThrTrpAlaGluThrMetGlyLeu 340345350
AATACCGCTGATACAGCAAGATTAAATGCCAATATTAGATATGTAAAT1104
AsnThrAlaAspThrAlaArgLeuAsnAlaAsnIleArgTyrValAsn 355360365
ACTGGGACGGCTCCAATCTACAACGTGTTACCAACGACTTCGTTAGTG1152
ThrGlyThrAlaProIleTyrAsnValLeuProThrThrSerLeuVal 370375380
TTAGGAAAAAATCAAACACTCGCGACAATTAAAGCTAAGGAAAACCAA1200
LeuGlyLysAsnGlnThrLeuAlaThrIleLysAlaLysGluAsnGln 385390395400
TTAAGTCAAATACTTGCACCTAATAATTATTATCCTTCTAAAAACTTG1248
LeuSerGlnIleLeuAlaProAsnAsnTyrTyrProSerLysAsnLeu 405410415
GCGCCAATCGCATTAAATGCACAAGACGATTTCAGTTCTACTCCAATT1296
AlaProIleAlaLeuAsnAlaGlnAspAspPheSerSerThrProIle 420425430
ACAATGAATTACAATCAATTTCTTGAGTTAGAAAAAACGAAACAATTA1344
ThrMetAsnTyrAsnGlnPheLeuGluLeuGluLysThrLysGlnLeu 435440445
AGATTAGATACGGATCAAGTATATGGGAATATAGCAACATACAATTTT1392
ArgLeuAspThrAspGlnValTyrGlyAsnIleAlaThrTyrAsnPhe 450455460
GAAAATGGAAGAGTGAGGGTGGATACAGGCTCGAACTGGAGTGAAGTG1440
GluAsnGlyArgValArgValAspThrGlySerAsnTrpSerGluVal 465470475480
TTACCGCAAATTCAAGAAACAACTGCACGTATCATTTTTAATGGAAAA1488
LeuProGlnIleGlnGluThrThrAlaArgIleIlePheAsnGlyLys 485490495
GATTTAAATCTGGTAGAAAGGCGGATAGCGGCGGTTAATCCTAGTGAT1536
AspLeuAsnLeuValGluArgArgIleAlaAlaValAsnProSerAsp 500505510
CCATTAGAAACGACTAAACCGGATATGACATTAAAAGAAGCCCTTAAA1584
ProLeuGluThrThrLysProAspMetThrLeuLysGluAlaLeuLys 515520525
ATAGCATTTGGATTTAACGAACCGAATGGAAACTTACAATATCAAGGG1632
IleAlaPheGlyPheAsnGluProAsnGlyAsnLeuGlnTyrGlnGly 530535540
AAAGACATAACCGAATTTGATTTTAATTTCGATCAACAAACATCTCAA1680
LysAspIleThrGluPheAspPheAsnPheAspGlnGlnThrSerGln 545550555560
AATATCAAGAATCAGTTAGCGGAATTAAACGCAACTAACATATATACT1728
AsnIleLysAsnGlnLeuAlaGluLeuAsnAlaThrAsnIleTyrThr 565570575
GTATTAGATAAAATCAAATTAAATGCAAAAATGAATATTTTAATAAGA1776
ValLeuAspLysIleLysLeuAsnAlaLysMetAsnIleLeuIleArg 580585590
GATAAACGTTTTCATTATGATAGAAATAACATAGCAGTTGGGGCGGAT1824
AspLysArgPheHisTyrAspArgAsnAsnIleAlaValGlyAlaAsp 595600605
GAGTCAGTAGTTAAGGAGGCTCATAGAGAAGTAATTAATTCGTCAACA1872
GluSerValValLysGluAlaHisArgGluValIleAsnSerSerThr 610615620
GAGGGATTATTGTTAAATATTGATAAGGATATAAGAAAAATATTATCA1920
GluGlyLeuLeuLeuAsnIleAspLysAspIleArgLysIleLeuSer 625630635640
GGTTATATTGTAGAAATTGAAGATACTGAAGGGCTTAAAGAAGTTATA1968
GlyTyrIleValGluIleGluAspThrGluGlyLeuLysGluValIle 645650655
AATGACAGATATGATATGTTGAATATTTCTAGTTTACGGCAAGATGGA2016
AsnAspArgTyrAspMetLeuAsnIleSerSerLeuArgGlnAspGly 660665670
AAAACATTTATAGATTTTAAAAAATATAATGATAAATTACCGTTATAT2064
LysThrPheIleAspPheLysLysTyrAsnAspLysLeuProLeuTyr 675680685
ATAAGTAATCCCAATTATAAGGTAAATGTATATGCTGTTACTAAAGAA2112
IleSerAsnProAsnTyrLysValAsnValTyrAlaValThrLysGlu 690695700
AACACTATTATTAATCCTAGTGAGAATGGGGATACTAGTACCAACGGG2160
AsnThrIleIleAsnProSerGluAsnGlyAspThrSerThrAsnGly 705710715720
ATCAAGAAAATTTTAAAGAAAGTGGTGCTGGGCAAAAAAGGGGATACA2208
IleLysLysIleLeuLysLysValValLeuGlyLysLysGlyAspThr 725730735
GTGGAACTGACCTGTACAGCTTCCCAGAAGAAGAGCATACAATTCCAC2256
ValGluLeuThrCysThrAlaSerGlnLysLysSerIleGlnPheHis 740745750
TGGAAAAACTCCAACCAGATAAAGATTCTGGGAAATCAGGGCTCCTTC2304
TrpLysAsnSerAsnGlnIleLysIleLeuGlyAsnGlnGlySerPhe 755760765
TTAACTAAAGGTCCATCCAAGCTGAATGATCGCGCTGACTCAAGAAGA2352
LeuThrLysGlyProSerLysLeuAsnAspArgAlaAspSerArgArg 770775780
AGCCTTTGGGACCAAGGAAACTTCCCCCTGATCATCAAGAATCTTAAG2400
SerLeuTrpAspGlnGlyAsnPheProLeuIleIleLysAsnLeuLys 785790795800
ATAGAAGACTCAGATACTTACATCTGTGAAGTGGAGGACCAGAAGGAG2448
IleGluAspSerAspThrTyrIleCysGluValGluAspGlnLysGlu 805810815
GAGGTGCAATTGCTAGTGTTCGGATTGACTGCCAACTCTGACACCCAC2496
GluValGlnLeuLeuValPheGlyLeuThrAlaAsnSerAspThrHis 820825830
CTGCTTCAGGGGCAGAGCCTGACCCTGACCTTGGAGAGCCCCCCTGGT2544
LeuLeuGlnGlyGlnSerLeuThrLeuThrLeuGluSerProProGly 835840845
AGTAGCCCCTCAGTGCAATGTAGGAGTCCAAGGGGTAAAAACATACAG2592
SerSerProSerValGlnCysArgSerProArgGlyLysAsnIleGln 850855860
GGGGGGAAGACCCTCTCCGTGTCTCAGCTGGAGCTCCAGGATAGTGGC2640
GlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly 865870875880
ACCTGGACATGCACTGTCTTGCAGAACCAGAAGAAGGTGGAGTTCAAA2688
ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLys 885890895
ATAGACATCGTGGTGCTAGCT2709 IleAspIleValValLeuAla 900 (2) INFORMATION
FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 903
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GluValLysGlnGluAsnArgLeuLeuAsnGluSerGluSerSerSer 151015
GlnGlyLeuLeuGlyTyrTyrPheSerAspLeuAsnPheGlnAlaPro 202530
MetValValThrSerSerThrThrGlyAspLeuSerIleProSerSer 354045
GluLeuGluAsnIleProSerGluAsnGlnTyrPheGlnSerAlaIle 505560
TrpSerGlyPheIleLysValLysLysSerAspGluTyrThrPheAla 65707580
ThrSerAlaAspAsnHisValThrMetTrpValAspAspGlnGluVal 859095
IleAsnLysAlaSerAsnSerAsnLysIleArgLeuGluLysGlyArg 100105110
LeuTyrGlnIleLysIleGlnTyrGlnArgGluAsnProThrGluLys 115120125
GlyLeuAspPheLysLeuTyrTrpThrAspSerGlnAsnLysLysGlu 130135140
ValIleSerSerAspAsnLeuGlnLeuProGluLeuLysGlnLysSer 145150155160
SerAsnSerArgLysLysArgSerThrSerAlaGlyProThrValPro 165170175
AspArgAspAsnAspGlyIleProAspSerLeuGluValGluGlyTyr 180185190
ThrValAspValLysAsnLysArgThrPheLeuSerProTrpIleSer 195200205
AsnIleHisGluLysLysGlyLeuThrLysTyrLysSerSerProGlu 210215220
LysTrpSerThrAlaSerAspProTyrSerAspPheGluLysValThr 225230235240
GlyArgIleAspLysAsnValSerProGluAlaArgHisProLeuVal 245250255
AlaAlaTyrProIleValHisValAspMetGluAsnIleIleLeuSer 260265270
LysAsnGluAspGlnSerThrGlnAsnThrAspSerGluThrArgThr 275280285
IleSerLysAsnThrSerThrSerArgThrHisThrSerGluValHis 290295300
GlyAsnAlaGluValHisAlaSerPhePheAspIleGlyGlySerVal 305310315320
SerAlaGlyPheSerAsnSerAsnSerSerThrValAlaIleAspHis 325330335
SerLeuSerLeuAlaGlyGluArgThrTrpAlaGluThrMetGlyLeu 340345350
AsnThrAlaAspThrAlaArgLeuAsnAlaAsnIleArgTyrValAsn 355360365
ThrGlyThrAlaProIleTyrAsnValLeuProThrThrSerLeuVal 370375380
LeuGlyLysAsnGlnThrLeuAlaThrIleLysAlaLysGluAsnGln 385390395400
LeuSerGlnIleLeuAlaProAsnAsnTyrTyrProSerLysAsnLeu 405410415
AlaProIleAlaLeuAsnAlaGlnAspAspPheSerSerThrProIle 420425430
ThrMetAsnTyrAsnGlnPheLeuGluLeuGluLysThrLysGlnLeu 435440445
ArgLeuAspThrAspGlnValTyrGlyAsnIleAlaThrTyrAsnPhe 450455460
GluAsnGlyArgValArgValAspThrGlySerAsnTrpSerGluVal 465470475480
LeuProGlnIleGlnGluThrThrAlaArgIleIlePheAsnGlyLys 485490495
AspLeuAsnLeuValGluArgArgIleAlaAlaValAsnProSerAsp 500505510
ProLeuGluThrThrLysProAspMetThrLeuLysGluAlaLeuLys 515520525
IleAlaPheGlyPheAsnGluProAsnGlyAsnLeuGlnTyrGlnGly 530535540
LysAspIleThrGluPheAspPheAsnPheAspGlnGlnThrSerGln 545550555560
AsnIleLysAsnGlnLeuAlaGluLeuAsnAlaThrAsnIleTyrThr 565570575
ValLeuAspLysIleLysLeuAsnAlaLysMetAsnIleLeuIleArg 580585590
AspLysArgPheHisTyrAspArgAsnAsnIleAlaValGlyAlaAsp 595600605
GluSerValValLysGluAlaHisArgGluValIleAsnSerSerThr 610615620
GluGlyLeuLeuLeuAsnIleAspLysAspIleArgLysIleLeuSer 625630635640
GlyTyrIleValGluIleGluAspThrGluGlyLeuLysGluValIle 645650655
AsnAspArgTyrAspMetLeuAsnIleSerSerLeuArgGlnAspGly 660665670
LysThrPheIleAspPheLysLysTyrAsnAspLysLeuProLeuTyr 675680685
IleSerAsnProAsnTyrLysValAsnValTyrAlaValThrLysGlu 690695700
AsnThrIleIleAsnProSerGluAsnGlyAspThrSerThrAsnGly 705710715720
IleLysLysIleLeuLysLysValValLeuGlyLysLysGlyAspThr 725730735
ValGluLeuThrCysThrAlaSerGlnLysLysSerIleGlnPheHis 740745750
TrpLysAsnSerAsnGlnIleLysIleLeuGlyAsnGlnGlySerPhe 755760765
LeuThrLysGlyProSerLysLeuAsnAspArgAlaAspSerArgArg 770775780
SerLeuTrpAspGlnGlyAsnPheProLeuIleIleLysAsnLeuLys 785790795800
IleGluAspSerAspThrTyrIleCysGluValGluAspGlnLysGlu 805810815
GluValGlnLeuLeuValPheGlyLeuThrAlaAsnSerAspThrHis 820825830
LeuLeuGlnGlyGlnSerLeuThrLeuThrLeuGluSerProProGly 835840845
SerSerProSerValGlnCysArgSerProArgGlyLysAsnIleGln 850855860
GlyGlyLysThrLeuSerValSerGlnLeuGluLeuGlnAspSerGly 865870875880
ThrTrpThrCysThrValLeuGlnAsnGlnLysLysValGluPheLys 885890895
IleAspIleValValLeuAla 900 (2) INFORMATION FOR SEQ ID NO:13: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix)
FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..8 (D) OTHER
INFORMATION: /label=PAHIV (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
SerGlnAsnTyrProValValGln 15 (2) INFORMATION FOR SEQ ID NO:14: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE:
internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis
(ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..12 (D) OTHER
INFORMATION: /label=PAHIV-1 (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:14: GlnValSerGlnAsnTyrProIleValGlnAsnIle 1510 (2) INFORMATION
FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12
amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM:
Bacillus anthracis (ix) FEATURE: (A) NAME/KEY: Peptide (B)
LOCATION: 1..12 (D) OTHER INFORMATION: /label=PAHIV-2 (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:15: AsnThrAlaThrIleMetMetGlnArgGlyAsnPhe
1510 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (vi)
ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix) FEATURE: (A)
NAME/KEY: Peptide (B) LOCATION: 1..12 (D) OTHER INFORMATION:
/label=PAHIV-3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
ThrValSerPheAsnPheProGlnIleThrLeuTrp 1510 (2) INFORMATION FOR SEQ
ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT
TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..13
(D) OTHER INFORMATION: /label=PAHIV-4 (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:17: GlyGlySerAlaPheAsnPheProIleValMetGlyGly 1510 (2)
INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..44 (D)
OTHER INFORMATION: /product="Primer 1A" (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:18: CGCAAGTATCACAAAATTATCCGATCGTGCAAAACATACTGCAG44
GlnValSerGlnAsnTyrProIleValGlnAsnIleLeuGln 1510 G45 (2) INFORMATION
FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GlnValSerGlnAsnTyrProIleValGlnAsnIleLeuGln 1510 (2) INFORMATION FOR
SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:
1..46 (D) OTHER INFORMATION: /product="Primer 1B" (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:20:
GTTCCTGCAGTATGTTTTGCACGATCGGATAATTTTGTGATACTTG46 (2) INFORMATION
FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..44 (D) OTHER
INFORMATION: /product="Primer 2A" (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:21: CGAACACTGCCACTATCATGATGCAACGTGGTAATTTTCTGCAG44
AsnThrAlaThrIleMetMetGlnArgGlyAsnPheLeuGln 1510 G45 (2) INFORMATION
FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14
amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
AsnThrAlaThrIleMetMetGlnArgGlyAsnPheLeuGln 1510 (2) INFORMATION FOR
SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:
1..46 (D) OTHER INFORMATION: /product="Primer 2B" (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:23:
GTCCCTGCAGAAAATTACCACGTTGCATCATGATAGTGGCAGTGTT46 (2) INFORMATION
FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..44 (D) OTHER
INFORMATION: /product="Primer 3A" (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:24: CGACTGTCTCTTTTAACTTCCCGCAAATCACGCTTTGGCTGCAG44
ThrValSerPheAsnPheProGlnIleThrLeuTrpLeuGln
1510 G45 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:25:
ThrValSerPheAsnPheProGlnIleThrLeuTrpLeuGln 1510 (2) INFORMATION FOR
SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:
1..46 (D) OTHER INFORMATION: /product="Primer 3B" (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:26:
GTCCCTGCAGCCAAAGCGTGATTTGCGGGAAGTTAAAAGAGACAGT46 (2) INFORMATION
FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus anthracis (ix)
FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 3..47 (D) OTHER
INFORMATION: /product="Primer 4A" (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:27: CGGGCGGTTCTGCCTTTAACTTCCCGATCGTCATGGGAGGTCTGCAG47
GlyGlySerAlaPheAsnPheProIleValMetGlyGlyLeuGln 151015 G48 (2)
INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:28: GlyGlySerAlaPheAsnPheProIleValMetGlyGlyLeuGln 151015 (2)
INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 49 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: (A)
ORGANISM: Bacillus anthracis (ix) FEATURE: (A) NAME/KEY:
misc_feature (B) LOCATION: 1..49 (D) OTHER INFORMATION:
/product="Primer 4B" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GTCCCTGCAGACCTCCCATGACGATCGGGAAGTTAAAGGCAGAACCGCC49 (2) INFORMATION
FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2160
base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus
anthracis (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..2157 (D)
OTHER INFORMATION: /product="PAHIV#2" (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:30: GAAGTTAAACAGGAGAACCGGTTATTAAATGAATCAGAATCAAGTTCC48
GluValLysGlnGluAsnArgLeuLeuAsnGluSerGluSerSerSer 151015
CAGGGGTTACTAGGATACTATTTTAGTGATTTGAATTTTCAAGCACCC96
GlnGlyLeuLeuGlyTyrTyrPheSerAspLeuAsnPheGlnAlaPro 202530
ATGGTGGTTACCTCTTCTACTACAGGGGATTTATCTATTCCTAGTTCT144
MetValValThrSerSerThrThrGlyAspLeuSerIleProSerSer 354045
GAGTTAGAAAATATTCCATCGGAAAACCAATATTTTCAATCTGCTATT192
GluLeuGluAsnIleProSerGluAsnGlnTyrPheGlnSerAlaIle 505560
TGGTCAGGATTTATCAAAGTTAAGAAGAGTGATGAATATACATTTGCT240
TrpSerGlyPheIleLysValLysLysSerAspGluTyrThrPheAla 65707580
ACTTCCGCTGATAATCATGTAACAATGTGGGTAGATGACCAAGAAGTG288
ThrSerAlaAspAsnHisValThrMetTrpValAspAspGlnGluVal 859095
ATTAATAAAGCTTCTAATTCTAACAAAATCAGATTAGAAAAAGGAAGA336
IleAsnLysAlaSerAsnSerAsnLysIleArgLeuGluLysGlyArg 100105110
TTATATCAAATAAAAATTCAATATCAACGAGAAAATCCTACTGAAAAA384
LeuTyrGlnIleLysIleGlnTyrGlnArgGluAsnProThrGluLys 115120125
GGATTGGATTTCAAGTTGTACTGGACCGATTCTCAAAATAAAAAAGAA432
GlyLeuAspPheLysLeuTyrTrpThrAspSerGlnAsnLysLysGlu 130135140
GTGATTTCTAGTGATAACTTACAATTGCCAGAATTAAAACAAAAATCT480
ValIleSerSerAspAsnLeuGlnLeuProGluLeuLysGlnLysSer 145150155160
TCGAACACTGCCACTATCATGATGCAACGTGGTAATTTTCTGCAGGGA528
SerAsnThrAlaThrIleMetMetGlnArgGlyAsnPheLeuGlnGly 165170175
CCTACGGTTCCAGACCGTGACAATGATGGAATCCCTGATTCATTAGAG576
ProThrValProAspArgAspAsnAspGlyIleProAspSerLeuGlu 180185190
GTAGAAGGATATACGGTTGATGTCAAAAATAAAAGAACTTTTCTTTCA624
ValGluGlyTyrThrValAspValLysAsnLysArgThrPheLeuSer 195200205
CCATGGATTTCTAATATTCATGAAAAGAAAGGATTAACCAAATATAAA672
ProTrpIleSerAsnIleHisGluLysLysGlyLeuThrLysTyrLys 210215220
TCATCTCCTGAAAAATGGAGCACGGCTTCTGATCCGTACAGTGATTTC720
SerSerProGluLysTrpSerThrAlaSerAspProTyrSerAspPhe 225230235240
GAAAAGGTTACAGGACGGATTGATAAGAATGTATCACCAGAGGCAAGA768
GluLysValThrGlyArgIleAspLysAsnValSerProGluAlaArg 245250255
CACCCCCTTGTGGCAGCTTATCCGATTGTACATGTAGATATGGAGAAT816
HisProLeuValAlaAlaTyrProIleValHisValAspMetGluAsn 260265270
ATTATTCTCTCAAAAAATGAGGATCAATCCACACAGAATACTGATAGT864
IleIleLeuSerLysAsnGluAspGlnSerThrGlnAsnThrAspSer 275280285
GAAACGAGAACAATAAGTAAAAATACTTCTACAAGTAGGACACATACT912
GluThrArgThrIleSerLysAsnThrSerThrSerArgThrHisThr 290295300
AGTGAAGTACATGGAAATGCAGAAGTGCATGCGTCGTTCTTTGATATT960
SerGluValHisGlyAsnAlaGluValHisAlaSerPhePheAspIle 305310315320
GGTGGGAGTGTATCTGCAGGATTTAGTAATTCGAATTCAAGTACGGTC1008
GlyGlySerValSerAlaGlyPheSerAsnSerAsnSerSerThrVal 325330335
GCAATTGATCATTCACTATCTCTAGCAGGGGAAAGAACTTGGGCTGAA1056
AlaIleAspHisSerLeuSerLeuAlaGlyGluArgThrTrpAlaGlu 340345350
ACAATGGGTTTAAATACCGCTGATACAGCAAGATTAAATGCCAATATT1104
ThrMetGlyLeuAsnThrAlaAspThrAlaArgLeuAsnAlaAsnIle 355360365
AGATATGTAAATACTGGGACGGCTCCAATCTACAACGTGTTACCAACG1152
ArgTyrValAsnThrGlyThrAlaProIleTyrAsnValLeuProThr 370375380
ACTTCGTTAGTGTTAGGAAAAAATCAAACACTCGCGACAATTAAAGCT1200
ThrSerLeuValLeuGlyLysAsnGlnThrLeuAlaThrIleLysAla 385390395400
AAGGAAAACCAATTAAGTCAAATACTTGCACCTAATAATTATTATCCT1248
LysGluAsnGlnLeuSerGlnIleLeuAlaProAsnAsnTyrTyrPro 405410415
TCTAAAAACTTGGCGCCAATCGCATTAAATGCACAAGACGATTTCAGT1296
SerLysAsnLeuAlaProIleAlaLeuAsnAlaGlnAspAspPheSer 420425430
TCTACTCCAATTACAATGAATTACGGGAATATAGCAACATACAATTTT1344
SerThrProIleThrMetAsnTyrGlyAsnIleAlaThrTyrAsnPhe 435440445
GAAAATGGAAGAGTGAGGGTGGATACAGGCTCGAACTGGAGTGAAGTG1392
GluAsnGlyArgValArgValAspThrGlySerAsnTrpSerGluVal 450455460
TTACCGCAAATTCAAGAAACAACTGCACGTATCATTTTTAATGGAAAA1440
LeuProGlnIleGlnGluThrThrAlaArgIleIlePheAsnGlyLys 465470475480
GATTTAAATCTGGTAGAAAGGCGGATAGCGGCGGTTAATCCTAGTGAT1488
AspLeuAsnLeuValGluArgArgIleAlaAlaValAsnProSerAsp 485490495
CCATTAGAAACGACTAAACCGGATATGACATTAAAAGAAGCCCTTAAA1536
ProLeuGluThrThrLysProAspMetThrLeuLysGluAlaLeuLys 500505510
ATAGCATTTGGATTTAACGAACCGAATGGAAACTTACAATATCAAGGG1584
IleAlaPheGlyPheAsnGluProAsnGlyAsnLeuGlnTyrGlnGly 515520525
AAAGACATAACCGAATTTGATTTTAATTTCGATCAACAAACATCTCAA1632
LysAspIleThrGluPheAspPheAsnPheAspGlnGlnThrSerGln 530535540
AATATCAAGAATCAGTTAGCGGAATTAAACGCAACTAACATATATACT1680
AsnIleLysAsnGlnLeuAlaGluLeuAsnAlaThrAsnIleTyrThr 545550555560
GTATTAGATAAAATCAAATTAAATGCAAAAATGAATATTTTAATAAGA1728
ValLeuAspLysIleLysLeuAsnAlaLysMetAsnIleLeuIleArg 565570575
GATAAACGTTTTCATTATGATAGAAATAACATAGCAGTTGGGGCGGAT1776
AspLysArgPheHisTyrAspArgAsnAsnIleAlaValGlyAlaAsp 580585590
GAGTCAGTAGTTAAGGAGGCTCATAGAGAAGTAATTAATTCGTCAACA1824
GluSerValValLysGluAlaHisArgGluValIleAsnSerSerThr 595600605
GAGGGATTATTGTTAAATATTGATAAGGATATAAGAAAAATATTATCA1872
GluGlyLeuLeuLeuAsnIleAspLysAspIleArgLysIleLeuSer 610615620
GGTTATATTGTAGAAATTGAAGATACTGAAGGGCTTAAAGAAGTTATA1920
GlyTyrIleValGluIleGluAspThrGluGlyLeuLysGluValIle 625630635640
AATGACAGATATGATATGTTGAATATTTCTAGTTTACGGCAAGATGGA1968
AsnAspArgTyrAspMetLeuAsnIleSerSerLeuArgGlnAspGly 645650655
AAAACATTTATAGATTTTAAAAAATATAATGATAAATTACCGTTATAT2016
LysThrPheIleAspPheLysLysTyrAsnAspLysLeuProLeuTyr 660665670
ATAAGTAATCCCAATTATAAGGTAAATGTATATGCTGTTACTAAAGAA2064
IleSerAsnProAsnTyrLysValAsnValTyrAlaValThrLysGlu 675680685
AACACTATTATTAATCCTAGTGAGAATGGGGATACTAGTACCAACGGG2112
AsnThrIleIleAsnProSerGluAsnGlyAspThrSerThrAsnGly 690695700
ATCAAGAAAATTTTAATCTTTTCTAAAAAAGGCTATGAGATAGGA2157
IleLysLysIleLeuIlePheSerLysLysGlyTyrGluIleGly 705710715 TAA2160 (2)
INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 719 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:31: GluValLysGlnGluAsnArgLeuLeuAsnGluSerGluSerSerSer 151015
GlnGlyLeuLeuGlyTyrTyrPheSerAspLeuAsnPheGlnAlaPro 202530
MetValValThrSerSerThrThrGlyAspLeuSerIleProSerSer 354045
GluLeuGluAsnIleProSerGluAsnGlnTyrPheGlnSerAlaIle 505560
TrpSerGlyPheIleLysValLysLysSerAspGluTyrThrPheAla 65707580
ThrSerAlaAspAsnHisValThrMetTrpValAspAspGlnGluVal 859095
IleAsnLysAlaSerAsnSerAsnLysIleArgLeuGluLysGlyArg 100105110
LeuTyrGlnIleLysIleGlnTyrGlnArgGluAsnProThrGluLys 115120125
GlyLeuAspPheLysLeuTyrTrpThrAspSerGlnAsnLysLysGlu 130135140
ValIleSerSerAspAsnLeuGlnLeuProGluLeuLysGlnLysSer 145150155160
SerAsnThrAlaThrIleMetMetGlnArgGlyAsnPheLeuGlnGly 165170175
ProThrValProAspArgAspAsnAspGlyIleProAspSerLeuGlu 180185190
ValGluGlyTyrThrValAspValLysAsnLysArgThrPheLeuSer 195200205
ProTrpIleSerAsnIleHisGluLysLysGlyLeuThrLysTyrLys 210215220
SerSerProGluLysTrpSerThrAlaSerAspProTyrSerAspPhe 225230235240
GluLysValThrGlyArgIleAspLysAsnValSerProGluAlaArg 245250255
HisProLeuValAlaAlaTyrProIleValHisValAspMetGluAsn 260265270
IleIleLeuSerLysAsnGluAspGlnSerThrGlnAsnThrAspSer 275280285
GluThrArgThrIleSerLysAsnThrSerThrSerArgThrHisThr 290295300
SerGluValHisGlyAsnAlaGluValHisAlaSerPhePheAspIle 305310315320
GlyGlySerValSerAlaGlyPheSerAsnSerAsnSerSerThrVal 325330335
AlaIleAspHisSerLeuSerLeuAlaGlyGluArgThrTrpAlaGlu 340345350
ThrMetGlyLeuAsnThrAlaAspThrAlaArgLeuAsnAlaAsnIle 355360365
ArgTyrValAsnThrGlyThrAlaProIleTyrAsnValLeuProThr 370375380
ThrSerLeuValLeuGlyLysAsnGlnThrLeuAlaThrIleLysAla 385390395400
LysGluAsnGlnLeuSerGlnIleLeuAlaProAsnAsnTyrTyrPro 405410415
SerLysAsnLeuAlaProIleAlaLeuAsnAlaGlnAspAspPheSer 420425430
SerThrProIleThrMetAsnTyrGlyAsnIleAlaThrTyrAsnPhe 435440445
GluAsnGlyArgValArgValAspThrGlySerAsnTrpSerGluVal 450455460
LeuProGlnIleGlnGluThrThrAlaArgIleIlePheAsnGlyLys 465470475480
AspLeuAsnLeuValGluArgArgIleAlaAlaValAsnProSerAsp 485490495
ProLeuGluThrThrLysProAspMetThrLeuLysGluAlaLeuLys 500505510
IleAlaPheGlyPheAsnGluProAsnGlyAsnLeuGlnTyrGlnGly 515520525
LysAspIleThrGluPheAspPheAsnPheAspGlnGlnThrSerGln 530535540
AsnIleLysAsnGlnLeuAlaGluLeuAsnAlaThrAsnIleTyrThr 545550555560
ValLeuAspLysIleLysLeuAsnAlaLysMetAsnIleLeuIleArg 565570575
AspLysArgPheHisTyrAspArgAsnAsnIleAlaValGlyAlaAsp 580585590
GluSerValValLysGluAlaHisArgGluValIleAsnSerSerThr 595600605
GluGlyLeuLeuLeuAsnIleAspLysAspIleArgLysIleLeuSer 610615620
GlyTyrIleValGluIleGluAspThrGluGlyLeuLysGluValIle 625630635640
AsnAspArgTyrAspMetLeuAsnIleSerSerLeuArgGlnAspGly 645650655
LysThrPheIleAspPheLysLysTyrAsnAspLysLeuProLeuTyr 660665670
IleSerAsnProAsnTyrLysValAsnValTyrAlaValThrLysGlu 675680685
AsnThrIleIleAsnProSerGluAsnGlyAspThrSerThrAsnGly 690695700
IleLysLysIleLeuIlePheSerLysLysGlyTyrGluIleGly 705710715 (2)
INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D)
TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:32: ArgLysLysArg (2) INFORMATION FOR SEQ ID
NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B)
TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
ArgGluAspLeuLys 15 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C)
STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:34: LeuAspGluArg 1 (2) INFORMATION
FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:35: ValAlaAlaTyrProIleValHisVal 15
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